bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription and decay of the lac messenger: role of an intergenic terminator
Authors: Murakawa G J , Kwan C , Yamashita J , Nierlich D P
Published: November 2016

Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.
Genetic transformation in Streptococcus pneumoniae: nucleotide sequence analysis shows comA, a gene required for competence induction, to be a member of the bacterial ATP-dependent transport protein family
Authors: Hui F M , Morrison D A
Published: November 2016

The complete nucleotide sequence of comA, a gene required for induction of competence for genetic transformation in Streptococcus pneumoniae, was determined by using plasmid DNA templates and synthetic oligonucleotide primers. The sequence contained a single large open reading frame, ORF1, of 2,151 bp. ORF1 was included within the comAB locus previously mapped genetically and accounted for 50% of its extent. The predicted molecular weight of the largest polypeptide encoded within ORF1, 80,290, coincided with that measured previously (77,000) for the product of in vitro transcription-translation of the cloned comA locus. A Shine-Dalgarno sequence (AAAGGAG, delta G = -14 kcal) lay immediately upstream of ORF1. A sequence (TTtAat-17 bp-TAaAAT) similar to the Escherichia coli sigma 70 promoter consensus was located 410 bp upstream of ORF1. The deduced protein sequence of ComA showed a very strong similarity to the E. coli hemolysin secretion protein, HlyB, and strong similarities to other members of the family of ATP-dependent transport proteins, including the mammalian multidrug resistance P-glycoprotein. These similarities suggest that ComA functions in the transport of some molecule, possibly pneumococcal competence factor itself.
Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium
Authors: Sugiyama T , Kido N , Komatsu T , Ohta M , Kato N
Published: November 2016

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.
Analysis of an mRNA exhibiting anomalous translational specificity
Authors: Vellanoweth R L , Rabinowitz J C
Published: November 2016

Gene 6 mRNA of Bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by Escherichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rare example of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by E. coli and B. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibited by moderate levels of KCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. This preferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing the general phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis. Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride was inhibitory at all concentrations tested.
Transcription of Clostridium thermocellum endoglucanase genes celF and celD
Authors: Mishra S , Béguin P , Aubert J P
Published: November 2016

Transcripts of the Clostridium thermocellum endoglucanase genes celF and celD, encoding endoglucanases F and D, respectively, were characterized. The size of the mRNAs was about 2.35 kb for celF and 2.1 kb for celD, indicating monocistronic transcription of both genes. A unique 5' end, located 218 bp upstream from the initiation codon, was found for celF mRNA. No convincing homology could be identified between the sequence upstream from the celF 5' end and other procaryotic promoters. Two 5' ends, located 124 and 294 bp upstream from the initiation codon, were mapped for celD mRNA. The -10 and the -35 sequences preceding the ATG-distal 5' end of celD mRNA were homologous to the consensus sequence of Bacillus subtilis sigma 43 promoters. The sequence upstream from the ATG-proximal 5' end had some similarity with the -10 sequence of B. subtilis sigma 28 promoters. During growth on cellobiose, the 5' end of celD transcripts was found predominantly at the -124 site during the late exponential phase but almost exclusively at the -294 site during the early stationary phase. The kinetics of appearance of celA, celC, celD, and celF mRNA was followed by dot blot analysis. Transcripts of celA, celD, and celF were detected during late exponential and early stationary phase. In contrast, the celC transcript was detected almost exclusively during early stationary phase. Since growth was limited by the availability of cellobiose, the results suggest that the genes are regulated by a mechanism analogous to catabolite repression.
Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120
Authors: Bergsland K J , Haselkorn R
Published: November 2016

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.
Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2
Authors: Goncharoff P , Saadi S , Chang C H , Saltman L H , Figurski D H
Published: November 2016

The kil loci (kilA, kilB, kilC, and kilE) of incompatibility group P (IncP), broad-host-range plasmid RK2 were originally detected by their potential lethality to Escherichia coli host cells. Expression of the kil determinants is controlled by different combinations of kor functions (korA, korB, korC, and korE). This system of regulated genes, known as the kil-kor regulon, includes trfA, which encodes the RK2 replication initiator. The functions of the kil loci are unknown, but their coregulation with an essential replication function suggests that they have a role in the maintenance or host range of RK2. In this study, we have determined the nucleotide sequence of a 3-kb segment of RK2 that encodes the entire kilA locus. The region encodes three genes, designated klaA, klaB, and klaC. The phage T7 RNA polymerase-dependent expression system was use to identify three polypeptide products. The estimated masses of klaA and klaB products were in reasonable agreement with the calculated molecular masses of 28,407 and 42,156 Da, respectively. The klaC product is calculated to be 32,380 Da, but the observed polypeptide exhibited an apparent mass of 28 kDa on sodium dodecyl sulfate-polyacrylamide gels. Mutants of klaC were used to confirm that initiation of translation of the observed product occurs at the first ATG in the klaC open reading frame. Hydrophobicity analysis indicated that the KlaA and KlaB polypeptides are likely to be soluble, whereas the KlaC polypeptide was predicted to have four potential membrane-spanning domains. The only recognizable promoter sequences in the kilA region were those of the kilA promoter located upstream of klaA and the promoter for the korA-korB operon located just downstream of a rho-independent terminatorlike sequence following klaC. The transcriptional start sites for these promoters were determined by primer extension. Using isogenic sets of plasmids with nonpolar mutations, we found that klaA, klaB, and klaC are each able to express a host-lethal (Kil+) phenotype in the absence of kor functions. Inactivation of the kilA promoter causes loss of the lethal phenotype, demonstrating that all three genes are expressed from the kilA promoter as a multicistronic operon. We investigated two other phenotypes that have been mapped to the kilA region of RK2 or the closely related IncP plasmids RP1 and RP4: inhibition of conjugal transfer of IncW plasmids (fwB) and resistance to potassium tellurite. The cloned kilA operon was found to express both phenotypes, even in the presence of korA and korB, whose functions are known to regulate the kilA promoter. In addition, mutant and complementation analyses showed that the kilA promoter and the products of all three kla genes are necessary for expression of both phenotypes. Therefore, host lethality, fertility inhibition, and tellurite resistance are all properties of the kilA operon. We discuss the possible role of the kilA operon for RK2.
Induction of manganese-superoxide dismutase by membrane-binding drugs in Escherichia coli
Authors: Zhang Q M , Yonei S
Published: November 2016

Treatment of exponentially growing cells of Escherichia coli with membrane-binding drugs such as chlorpromazine (CPZ) and procaine resulted in an induction of manganese-superoxide dismutase (Mn-SOD). A slight decrease was observed in the amount of Fe-SOD. The induction of Mn-SOD required de novo synthesis of this enzyme, since it was suppressed by rifampin. The treatment did not cause the induction of Mn-SOD when performed under anaerobic conditions. In E. coli cells with a sodA-lacZ operon fusion, CPZ and procaine induced beta-galactosidase in the presence of oxygen, whereas it was not expressed and was not induced by CPZ and procaine under anaerobic conditions. Although CPZ reduced the ability of cell suspensions to take up oxygen, it increased the cyanide-resistant fraction of the total respiration. Therefore, it appeared likely that the induction of the sodA gene was a response to an increase in superoxide radical production mediated by these membrane-binding drugs in E. coli cells, possibly by disruption of the electron transport systems in the cell membranes.
Early recognition in the Rhizobium meliloti-alfalfa symbiosis: root exudate factor stimulates root adsorption of homologous rhizobia
Authors: Wall L G , Favelukes G
Published: November 2016

Adsorption of Rhizobium meliloti to alfalfa roots before their infection and nodule formation shows the specificity of the symbiotic association (G. Caetano-Anollés and G. Favelukes, Appl. Environ. Microbiol. 52:377-382, 1986). The time course of specific adsorption of R. meliloti (10(3) to 10(4) cells per ml) to roots shows an initial lag period of 3 h, suggesting that either or both symbionts must become conditioned for the adsorption process. Preincubation of R. meliloti L5-30 for 3 h with dialyzed alfalfa root exudate (RE) markedly increased early adsorption of rhizobia to alfalfa roots. The activity in RE was linked to a nondialyzable, thermolabile, trypsin-sensitive factor(s), very different from the root-exuded flavonoid compounds also involved in early Rhizobium-legume interactions. The lack of activity in the RE from plants grown in 5 mM NO3- suggested its negative regulation by the nitrogen nutritional status of the plant. Preincubation of R. meliloti with heterologous clover RE did not stimulate adsorption of rhizobial cells to roots. A short pretreatment of RE with homologous (but not heterologous) strains eliminated the stimulatory activity from solution. The stimulation of adsorption of R. meliloti to alfalfa roots was strongly dependent on the growth phase of the rhizobia, being greater at the late exponential stage. Nevertheless, the capacity of R. meliloti L5-30 to eliminate from solution the stimulatory activity in RE appeared to be constitutive in the rhizobia. The low concentration of rhizobial cells used in these experiments was critical to detect the stimulation of adsorption. The early interaction of spontaneously released alfalfa root macromolecular factor(s) and free-living R. meliloti, which shows the specificity and regulatory properties characteristic of infection and nodulation, would be an initial recognition event in the rhizosphere which triggers the process of symbiotic association.
Evidence for two promoters for the cytochrome c2 gene (cycA) of Rhodobacter sphaeroides
Authors: MacGregor B J , Donohue T J
Published: November 2016

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a periplasmic heme protein, encoded by cycA, that is required for photosynthetic growth and for one branch of the aerobic electron transport chain. cycA mRNA and cyt c2 are more abundant photosynthetically than aerobically. We report here that there are four cycA transcripts by high-resolution Northern (RNA) blot analysis, and we have mapped 10 5' ends by primer extension. Complementation of a cycA null mutant shows that there are at least two cycA promoters: one within 89 bp upstream of the translation initiation codon for a transcript beginning at -28, and at least one within 484 bp upstream for the remaining nine 5' ends. The 5' ends at -28 and -137 are more abundant in aerobically grown cells, while those at -38, -155, -250, and -300 are more abundant photosynthetically. DNA sequences with homology to the Escherichia coli sigma 70 consensus promoter sequence precede the 5' ends at -28 and -274, and there is weak homology upstream of the -82 and -250 ends.
cis-acting ompF mutations that result in OmpR-dependent constitutive expression
Authors: Slauch J M , Silhavy T J
Published: November 2016

OmpR and EnvZ differentially control the transcription of the major outer membrane porin genes, ompF and ompC, in Escherichia coli in response to the osmolarity of the medium. We have previously provided evidence that OmpR works both positively and negatively at the ompF promoter to give the characteristic switch from OmpF to OmpC production with increasing osmolarity. Here, we describe the isolation of cis-acting ompF mutations that affect negative regulation by OmpR by affecting the three-dimensional structure of the promoter region as measured by agarose gel mobility. These results further clarify the mechanism by which OmpR negatively regulates ompF expression, suggesting a model in which OmpR forms a repressive loop in the ompF promoter region.
Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism
Authors: Priefert H , Hein S , Krüger N , Zeh K , Schmidt B , Steinbüchel A
Published: November 2016

Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude extracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of alpha and beta subunits (Mrs, 35,500 and 36,000, respectively), and a tetrameric alpha 2 beta 2 structure was most likely for the native protein. The molecular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were determined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the alpha and beta subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, acoB, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, and the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together with the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading frame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial sigma 54-dependent promoter consensus sequence. The amino acid sequences deduced from acoA and acoB for the alpha subunit (Mr, 35,243) and the beta subunit (Mr, 35,788) exhibited significant homologies to the primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whereas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltransferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.
Phylogenetic relationships among Frankia genomic species determined by use of amplified 16S rDNA sequences
Authors: Nazaret S , Cournoyer B , Normand P , Simonet P
Published: November 2016

Actinomycetes of the genus Frankia establish a nitrogen-fixing symbiosis with a large number of woody dicotyledonous plants. Hundreds of strains isolated from various actinorhizal plants growing in different geographical areas have recently been classified into at least nine genomic species by use of the DNA-DNA hybridization technique (M.P. Fernandez, H. Meugnier, P.A.D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989). A protocol based on the amplification and sequencing of 16S ribosomal DNA segments was used to classify and estimate the phylogenetic relationships among eight different genomic species. A good correlation was established between the grouping of strains according to their 16S ribosomal DNA sequence homology and that based on total DNA homology, since most genomic species could be characterized by a specific sequence. The phylogenetic tree showed that strains belonging to the Alnus infectivity group are closely related to strains belonging to the Casuarina infectivity group and that strains of these two infectivity groups are well separated from strains of the Elaeagnus infectivity group, which also includes atypical strains isolated from the Casuarina group. This phylogenetic analysis was also very efficient for classifying previously unclassified pure cultures or unisolatable strains by using total DNA extracted directly from nodules.
Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae
Authors: Kinscherf T G , Coleman R H , Barta T M , Willis D K
Published: November 2016

Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the beta-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked Tn5 insertions and retained their resistance to the active toxin form, tabtoxnine-beta-lactam (T beta L). The wild-type DNA corresponding to the mutated region was cloned and found to restore the Tn5 mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10(-3)/CFU) in BR2. The Tox- deletion mutants also lost resistance to tabtoxinine-beta-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox- derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease.
Cloning, nucleotide sequence, and enzymatic characterization of an alpha-amylase from the ruminal bacterium Butyrivibrio fibrisolvens H17c
Authors: Rumbak E , Rawlings D E , Lindsey G G , Woods D R
Published: November 2016

A Butyrivibrio fibrisolvens amylase gene was cloned and expressed by using its own promoter on the recombinant plasmid pBAMY100 in Escherichia coli. The amylase gene consisted of an open reading frame of 2,931 bp encoding a protein of 976 amino acids with a calculated Mr of 106,964. In E. coli(pBAMY100), more than 86% of the active amylase was located in the periplasm, and TnphoA fusion experiments showed that the enzyme had a functional signal peptide. The B. fibrisolvens amylase is a calcium metalloenzyme, and three conserved putative calcium-binding residues were identified. The amylase showed high sequence homology with other alpha-amylases in the three highly conserved regions which constitute the active centers. These and other conserved regions were located in the N-terminal half, and no similarity with any other amylase was detected in the remainder of the protein. Deletion of approximately 40% of the C-terminal portion of the amylase did not result in loss of amylolytic activity. The B. fibrisolvens amylase was identified as an endo-alpha-amylase by hydrolysis of the Phadebas amylase substrate, hydrolysis of gamma-cyclodextrin to maltotriose, maltose, and glucose and the characteristic shape of the blue value and reducing sugar curves. Maltotriose was the major initial hydrolysis product from starch, although extended incubation resulted in its hydrolysis to maltose and glucose.
Nucleotide sequence and characterization of the gene for secreted alkaline phosphatase from Lysobacter enzymogenes
Authors: Au S , Roy K L , von Tigerstrom R G
Published: November 2016

Lysobacter enzymogenes produces an alkaline phosphatase which is secreted into the medium. The gene for the enzyme (phoA) was isolated from a recombinant lambda library. It was identified within a 4.4-kb EcoRI-BamH1 fragment, and its sequence was determined by the chain termination method. The structural gene consists of an open reading frame which encodes a 539-amino-acid protein with a 29-residue signal sequence, followed by a 119-residue propeptide, the 281-residue mature phosphatase, and a 110-residue carboxy-terminal domain. The roles of the propeptide and the carboxy-terminal peptide remain to be determined. A molecular weight of 30,000 was determined for the mature enzyme from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence was compared with sequences available in the current protein data base, and a region of the sequence was found to show considerable homology with sequences in mammalian type 5 iron-containing purple acid phosphatases.
Analysis of transcriptional control of the gerD spore germination gene of Bacillus subtilis 168
Authors: Kemp E H , Sammons R L , Moir A , Sun D , Setlow P
Published: November 2016

The gerD locus of Bacillus subtilis comprises a single gene whose function is essential for the germination of B. subtilis spores in media containing asparagine, glucose, and fructose. The expression of gerD has been characterized by using a chromosomal lacZ fusion to the gerD promoter. The promoter is switched on at the same time as the synthesis of glucose dehydrogenase, 2.5 h after sporulation has been initiated in the developing forespore. The gerD gene is not expressed in spoIIB or spoIIIA, -IIIB, -EIII, -FIII, or -IIIG mutants, but it is expressed in spoIIIC and -IIID and spoIVA mutant backgrounds. The in vivo transcriptional start point of the gene has been mapped by primer extension analysis, and sequences upstream from the start point show considerable homology with the promoter consensus sequences recognized by RNA polymerase containing the forespore-specific sigma factor sigma G (E sigma G). gerD is transcribed in vitro by E sigma G with a similar if not identical start point to that found in vivo, and expression of the gene can be rapidly induced in vegetative cells following the induction of sigma G synthesis. These results indicate that gerD is another member of the sigma G regulon, which includes a number of genes expressed only in the forespore compartment of sporulating cells of B. subtilis.
Genetic and physical analyses of the growth rate-dependent regulation of Escherichia coli zwf expression
Authors: Rowley D L , Pease A J , Wolf R E
Published: November 2016

Growth rate-dependent regulation of the level of Escherichia coli glucose 6-phosphate dehydrogenase, encoded by zwf, and 6-phosphogluconate dehydrogenase, encoded by gnd, is similar during steady-state growth and after nutritional upshifts. To determine whether the mechanism regulating zwf expression is like that of gnd, which involves a site of posttranscriptional control located within the structural gene, we prepared and analyzed a set of zwf-lacZ protein fusions in which the fusion joints are distributed across the glucose 6-phosphate dehydrogenase coding sequence. Expression of beta-galactosidase from the protein fusions was as growth rate dependent as that of glucose 6-phosphate dehydrogenase itself, indicating that regulation does not involve an internal regulatory region. The level of beta-galactosidase in zwf-lac operon fusion strains and the level of zwf mRNA from a wild-type strain increased with increasing growth rate, which suggests that growth rate control is exerted on the mRNA level. The half-life of the zwf mRNA mass was 3.0 min during growth on glucose and 3.4 min during growth on acetate. Thus, zwf transcription appears to be the target for growth rate control of the glucose 6-phosphate dehydrogenase level.
Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO
Authors: Wolff J A , MacGregor C H , Eisenberg R C , Phibbs P V
Published: November 2016

Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.
Rifampin-resistant replication of pBR322 derivatives in Escherichia coli cells induced for the SOS response
Authors: Magee T R , Kogoma T
Published: November 2016

Replication of plasmid pBR322 in Escherichia coli cells normally requires RNA synthesis and thus is sensitive to rifampin, an inhibitor of RNA polymerase. In cells induced for the SOS response, however, derivatives of pBR322 were found to replicate in the presence of rifampin. This rifampin-resistant replication of pBR322 requires the insertion of certain sequences of DNA. The replication depends on recF+ and DNA polymerase I.
The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation
Authors: Rosengarten R , Wise K S
Published: November 2016

Isogenic populations of Mycoplasma hyorhinis undergo in vitro high-frequency phase variation in the expression of surface lipoproteins; these products also vary markedly in size through changes in periodic protein structure (R. Rosengarten and K.S. Wise, Science 247:315-318, 1990). In this report, we rigorously define three distinct translation products comprising the Vlp (variable lipoprotein) system of M. hyorhinis SK76 and establish parameters of Vlp structural diversity and expression that distinguish the Vlp system from previously described examples of antigenic variation. VlpA, VlpB, and VlpC are prominent amphiphilic membrane lipoproteins characterized by detergent-phase fractionation and metabolic labeling with [35S]cysteine and [3H]palmitate. VlpA is distinguished from VlpB and VlpC by its selective labeling with [35S]methionine; VlpB and VlpC are distinguished by specific epitopes defined by surface-binding monoclonal antibodies (MAbs); a third MAb defines a surface epitope shared by VlpB and VlpC (but absent from VlpA). Each Vlp displays 12 to 30 spontaneous size variant forms comprising a periodic ladder that could also be generated by partial trypsin digestion of individual Vlp size variants. Different periodic intervals within VlpB and VlpC further distinguish these two products structurally. Mycoplasma colony opacity correlates inversely with Vlp size. Each Vlp undergoes independent, oscillating high-frequency phase variation in isogenic populations and can be expressed individually or concomitantly with other Vlps in a noncoordinate manner. All seven possible combinations of these three products were observed; however, no variants were found that lacked a Vlp. High-frequency size variation of each Vlp superimposed on combinatorial diversity in Vlp expression yields greater than 10(4) possible structurally distinct Vlp mosaics, of which 104 were documented along with 24 of 42 possible transitions among the seven Vlp combinations. In addition to these features, VlpA, VlpB, and VlpC were specifically recognized by serum antibodies from swine with experimental M. hyorhinis SK76-induced arthritis, indicating expression and immunogenicity of Vlps in the natural host. The structure and variation of Vlps and their known involvement in MAb-mediated modulation of mycoplasma-infected host cell properties and mycoplasma killing are discussed in relation to the surface architecture and adaptive potential of the wall-less mycoplasmas.
Replacement recombination in Lactococcus lactis
Authors: Leenhouts K J , Kok J , Venema G
Published: November 2016

In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP). The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence. Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis. In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L. lactis chromosome. The other transformants in which the pepXP gene had not been inactivated carried a Campbell-type integrated copy of the plasmid. Loss of part of the Campbell-type integrated plasmid via recombination between 1.6-kb nontandem repeats occurred with low frequencies that varied between less than 2.8 x 10(-6) and 8.5 x 10(-6), producing cells with a chromosomal structure like that of cells in which replacement recombination had taken place.
Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data
Authors: Kordel M , Hofmann B , Schomburg D , Schmid R D
Published: November 2016

A procedure for the purification of a very hydrophobic lipase from Pseudomonas sp. strain ATCC 21808 was elaborated by avoiding the use of long-chain detergents in view of subsequent crystallization of the enzyme. The purification procedure included chromatography on Q-Sepharose in the presence of n-octyl-beta-D-glucopyranoside, Ca2+ precipitation of fatty acids, and Octyl-Sepharose chromatography. The enzyme was purified 260-fold to a yield of 35% and a specific activity of 3,300 U/mg. The molecular weight was determined as 35,000; a polyacrylamide gel under nondenaturing conditions revealed a band at 110,000, and the isoelectric point proved to be at 4.5 to 4.6. The lipase crystallized with different salts and ethylene glycol polymers in the presence of n-octyl-beta-D-glucopyranoside and one alkyloligooxyethylene compound (CxEy) in the range from C5E2 to C8E4. The crystals diffract to a resolution of about 0.25 nm. Precession photographs revealed that they belong to space group C2 with lattice constants of a = 9.27 nm, b = 4.74 nm, c = 8.65 nm, and beta = 122.3 degrees, indicating a cell content of one molecule per asymmetric unit of the crystal. In hydrolysis of triglycerides, the lipase showed substrate specificity for saturated fatty acids from C6 to C12 and unsaturated long-chain fatty acids. Monoglycerides were hydrolyzed very slowly. The N-terminal sequence is identical to that of the lipase from Pseudomonas cepacia. Treatment with diethyl-p-nitrophenylphosphate affected the activities toward triolein and p-nitrophenylacetate to the same extent and with the same velocity.
The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system
Authors: Reidl J , Boos W
Published: November 2016

Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes. A second site mutation in malI abolishes the constitutive expression. The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J. Reidl, K. Römisch, M. Ehrmann, and W. Boos, J. Bacteriol. 171:4888-4899, 1989). We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY. MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE). When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose. Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX. MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion. We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system. The second gene in the operon, malY, encodes a protein of 43,500 daltons. Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences. The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant. The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose. This is not the case in a malT(Con) strain that expresses the mal genes constitutively. We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis.
Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product
Authors: Wilson S , Drew R
Published: November 2016

Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.
In situ studies on incorporation of nucleic acid precursors into Chlamydia trachomatis DNA
Authors: McClarty G , Tipples G
Published: November 2016

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates. The purpose of the present study was to determine whether Chlamydia trachomatis obtains deoxyribonucleotides from the host cell. The study was aided by the finding that host and parasite DNA synthesis activity could be distinguished by their differing sensitivities to aphidicolin and norfloxacin. Results from isotope incorporation experiments indicated that any nucleobase or ribonucleoside that could serve as a precursor for host DNA synthesis could also be utilized by C. trachomatis for DNA replication. C. trachomatis utilized only those precursors which the host cell converted to the nucleotide level. Pyrimidine deoxyribonucleotides were efficient precursors for host DNA synthesis; however, they were not used by C. trachomatis. On the other hand, purine deoxyribonucleosides are rapidly catabolized by host cells, it is necessary to regulate their metabolism to determine whether they serve as direct precursors for C. trachomatis DNA synthesis. This was partially achieved by using a hypoxanthine-guanine phosphoribosyltransferase-negative cell line and using deoxycoformycin and 8-aminoguanosine as inhibitors of (deoxy)adenosine deaminase and purine nucleoside phosphorylase, respectively. The results indicated that purine deoxyribonucleosides are efficiently utilized for host cell DNA synthesis even if degradation pathways are inhibited and salvage to ribonucleotides is minimized. In sharp contrast, the purine deoxyribonucleosides were utilized by C. trachomatis as precursors for DNA synthesis only when host catabolic pathways and salvage reactions were intact. High-pressure liquid chromatographic analysis of nucleotide pools extracted from host cells pulsed with radiolabeled precursors suggests that infected cells transport and phosphorylate all deoxynucleosides as effectively as mock-infected control cultures. In aggregate, these results show that chlamydiae do not take up deoxyribonucleotides from the host cells.
Genetic evidence for interaction between the CheW and Tsr proteins during chemoreceptor signaling by Escherichia coli
Authors: Liu J D , Parkinson J S
Published: November 2016

This study presents two lines of genetic evidence consistent with the premise that CheW, a cytoplasmic component of the chemotactic signaling system of Escherichia coli, interacts directly with Tsr, the membrane-bound serine chemoreceptor. (i) We demonstrated phenotypic suppression between 10 missense mutant CheW proteins and six missense mutant Tsr proteins. Most of these mutant proteins had leaky chemotaxis defects and were partially dominant, implying relatively minor functional alterations. Their suppression pattern was allele specific, suggesting that the mutant proteins have compensatory conformational changes at sites of interactive contact. (ii) We isolated five partially dominant CheW mutations and found that four of them were similar or identical to the suppressible CheW mutant proteins. This implies that there are only a few ways in which CheW function can be altered to produce dominant defects and that dominance is mediated through interactions of CheW with Tsr. The amino acid replacements in these mutant proteins were inferred from their DNA sequence changes. The CheW mutations were located in five regularly spaced clusters in the first two-thirds of the protein. The Tsr mutations were located in a highly conserved region in the middle of the cytoplasmic signaling domain. The hydrophobic moments, overall hydrophobicities, and predicted secondary structures of the mutant segments were consistent with the possibility that they are located at the surface of the CheW and Tsr molecules and represent the contact sites between these two proteins.
Evidence for multiple terminal oxidases, including cytochrome d, in facultatively alkaliphilic Bacillus firmus OF4
Authors: Hicks D B , Plass R J , Quirk P G
Published: November 2016

The terminal oxidase content of Bacillus firmus OF4, a facultative alkaliphile that grows well over the pH range of 7.5 to 10.5, was studied by difference spectroscopy. Evidence was found for three terminal oxidases under different growth conditions. The growth pH and the stage of growth profoundly affected the expression of one of the oxidases, cytochrome d. The other two oxidases, cytochrome caa3 and cytochrome o, were expressed under all growth conditions tested, although the levels of both, especially cytochrome caa3, were higher at more alkaline pH (P.G. Quirk, A.A. Guffanti, R.J. Plass, S. Clejan, and T.A. Krulwich, Biochim. Biophys. Acta, in press). These latter oxidases were identified in everted membrane vesicles by reduced-versus-oxidized difference spectra (absorption maximum at 600 nm for cytochrome caa3) and CO-reduced-versus-reduced difference spectra (absorption maxima at 574 and 414 nm for cytochrome o). All three terminal oxidases were solubilized from everted membranes and partially purified. The difference spectra of the solubilized, partially purified cytochrome caa3 and cytochrome o complexes were consistent with these assignments. Cytochrome d, which has not been identified in a Bacillus species before, was tentatively assigned on the basis of its absorption maxima at 622 and 630 nm in reduced-versus-oxidized and CO-reduced-versus-reduced difference spectra, respectively, resembling the maxima exhibited by the complex found in Escherichia coli. The B. firmus OF4 cytochrome d was reducible by NADH but not by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine in everted membrane vesicles. Cytochrome d was expressed under two conditions: in cells growing exponentially at pH 7.5 (but not at pH 10.5) and in cells stationary phase at either pH 7.5 or 10.5. Protein immunoblots with antibodies against subunit I of the E. coli cytochrome d complex reacted only with membrane vesicles that contained spectrally identifiable cytochrome d. Additional evidence that this B. firmus OF4 cytochrome is related to the E. coli complex was obtained with a solubilized, partially purified fraction of cytochrome d that also reacted with antibodies against the subunits of the E. coli cytochrome d.
Mutational analysis of repression and activation of the tyrP gene in Escherichia coli
Authors: Andrews A E , Lawley B , Pittard A J
Published: November 2016

In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters. We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters. In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine. Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein. Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation. In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site.