bluuebunny/crossref_metadata_2025 huggingface dataset:
The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene
Authors: Shadel G S , Baldwin T O
Published: November 2016

Regulation of the genes required for bioluminescence in the marine bacterium Vibrio fischeri (the lux regulon) is a complex process requiring coordination of several systems. The primary level of regulation is mediated by a positive regulatory protein, LuxR, and a small diffusible molecule, N-(3-oxo-hexanoyl)-homoserine lactone, termed autoinducer. Transcription of the luxR gene, which encodes the regulatory protein, is positively regulated by the cyclic AMP-CAP system. The lux regulon of V. fischeri consists of two divergently transcribed operons designated operonL and operonR. Transcription of the rightward operon (operonR; luxICDABE), consisting of the genes required for autoinducer synthesis (luxI) and light production (luxCDABE), is activated by LuxR in an autoinducer-dependent fashion. The leftward operon (operonL) consists of a single known gene, luxR. The LuxR protein has also been shown to decrease transcription of operonL through an autoinducer-dependent mechanism, thereby negatively regulating its own synthesis. In this paper we demonstrate that the autoinducer-dependent repression of operonL transcription requires not only LuxR but also DNA sequences within operonR which occur upstream of the promoter for operonL. In the absence of these DNA sequences, the LuxR protein causes an autoinducer-dependent activation of transcription of operonL. The lux operator, located in the control region between the two operons, was required for both the positive and negative autoinducer-dependent responses. By titration of high levels of LuxR supplied in trans with synthetic autoinducer, we found that low levels of autoinducer could elicit a positive response even in the presence of the negative-acting DNA sequences, while higher levels of autoinducer resulted in a negative response. Without these DNA sequences in operonR, LuxR and autoinducer stimulated transcription regardless of the level of autoinducer. These results suggest that a switch between stimulation and repression of operonL transcription is mediated by the levels of the LuxR-autoinducer complex, which in these experiments reflects the level of autoinducer in the growth medium.
Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli
Authors: Brissette R E , Tsung K L , Inouye M
Published: November 2016

Phosphorylation of OmpR, a transcription activator for ompF and ompC expression, is essential for its function and has been shown to be mediated in vitro by EnvZ, a transmembrane sensory receptor protein. On the basis of the three-dimensional structure of CheY which has an extensive sequence similarity with OmpR, three aspartic residues, D11, D12, and D55, of OmpR are considered to form a triacidic pocket serving as the phosphorylation center. When these aspartic acid residues were replaced with asparagine (D11N) or glutamine (D12Q and D55Q), ompF and ompC expression was almost completely blocked. Two pseudorevertants of the D11N mutation were isolated: one of them is a mutation in EnvZ (G240E), and the other is a mutation in OmpR (S48F). The envZ mutation (G240E) by itself was found to confer a phenotype very similar to that of the well known envZ11 mutation (T247R), suggesting that EnvZ (G240E) is an elevated kinase for OmpR. Consistent with this notion, EnvZ (T247R) was also able to suppress the D11N mutation in OmpR. An in vitro phosphorylation study showed that while the wild-type OmpR was phosphorylated by EnvZ, the D11N OmpR was not. These results suggest that the D11N mutation alters OmpR conformation in such a way that OmpR is very poorly phosphorylated by EnvZ. On the basis of the in vivo and in vitro analysis, the mechanisms by which the G240E mutation in EnvZ and the S48F mutation in OmpR suppress the D11N mutation in OmpR are discussed.
Glycosylation and structure of the yeast MF alpha 1 alpha-factor precursor is important for efficient transport through the secretory pathway
Authors: Caplan S , Green R , Rocco J , Kurjan J
Published: November 2016

The MF alpha 1 gene encodes a precursor, prepro-alpha-factor, that undergoes several proteolytic processing steps within the classical secretory pathway to produce the mature peptide pheromone, alpha-factor. To investigate the role of structural features of the MF alpha 1 precursor in alpha-factor production, we analyzed the effect of mf alpha 1 mutations that alter precursor structure in a number of ways. These mutations resulted in decreased alpha-factor secretion and intracellular accumulation of pro-alpha-factor. With the exception of the mutant lacking all three N glycosylation sites, the pro-alpha-factor forms that accumulated were core glycosylated but had not yet undergone the addition of outer chain carbohydrate. The delay, therefore, occurred at a step prior to the first proteolytic processing step involved in maturation of the precursor and was probably due to inefficient endoplasmic reticulum-to-Golgi transport. Elimination of all three N-glycosylation sites caused a delay in disappearance of intracellular precursor, and alpha-factor secretion was also slowed. These data indicate that N glycosylation is important but not essential for transport of the precursor through the secretory pathway. The decreased alpha-factor secretion and increased precursor accumulation seen with many different structural changes of pro-alpha-factor indicate that the secretory pathway is extremely sensitive to changes in precursor structure. This sensitivity could cause inefficient secretion of heterologous proteins and hybrids between MF alpha 1 and heterologous proteins in yeast cells.
Direct proof of a "more-than-single-layered" peptidoglycan architecture of Escherichia coli W7: a neutron small-angle scattering study
Authors: Labischinski H , Goodell E W , Goodell A , Hochberg M L
Published: November 2016

A neutron small-angle scattering study was performed to determine the thickness and the scattering density profile of isolated peptidoglycan sacculi of Escherichia coli W7 in aqueous suspension (D2O). The maximum thickness (7 +/- 0.5 nm) of the sacculus from the exponential-phase cells was large enough to suggest the existence of a more-than-single-layered architecture. The experimental density profile across the thickness of the sacculus did not allow an unambiguous differentiation between a single-layered architecture characterized by completely extended peptide side chains projecting from the sugar strands or, alternatively, a partially triple layered structure. To resolve this ambiguity, sacculi were labeled with deuterated wall peptides. Comparison of the two experimental profiles indicated that the sacculus is more than single layered across its surface, with about 75 to 80% of its surface single layered and 20 to 25% triple layered.
Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene
Authors: Ullstrom C A , Siehnel R , Woodruff W , Steinbach S , Hancock R E
Published: November 2016

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.
Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus
Authors: Heyne R I , de Vrij W , Crielaard W , Konings W N
Published: November 2016

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.
Characterization of vanadate-dependent NADH oxidation stimulated by Saccharomyces cerevisiae plasma membranes
Authors: Minasi L A , Willsky G R
Published: November 2016

Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae. This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction. Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin. The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein. The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH. These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation. The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate. 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0. In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride. Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.
Isolation and analysis of dominant secA mutations in Escherichia coli
Authors: Jarosik G P , Oliver D B
Published: November 2016

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.
Osmotic signal transduction to proU is independent of DNA supercoiling in Escherichia coli
Authors: Ramirez R M , Villarejo M
Published: November 2016

proU expression has been proposed to form part of a general stress response that is regulated by increased negative DNA supercoiling brought about by environmental signals such as osmotic or anaerobic stress (N. Ni Bhriain, C. J. Dorman, and C. F. Higgins, Mol. Microbiol. 3:933-944, 1989). However, we find that although proU-containing plasmids derived from cells grown in media of elevated osmolarity were more supercoiled than plasmids from cells grown in standard media, they did not activate proU expression in vitro. The gyrA96 mutation and anaerobic conditions are known to affect DNA supercoiling but did not alter proU expression. Finally, the gyrase inhibitors coumermycin and novobiocin did not reduce in vitro proU expression. Therefore, this evidence rules out regulation by changes in DNA superhelicity for proU in Escherichia coli.
Peptidoglycan precursor from Fusobacterium nucleatum contains lanthionine
Authors: Fredriksen A , Vasstrand E N , Jensen H B
Published: November 2016

Fusobacterium nucleatum was grown in the presence of [14C]UDP. By means of sequential precipitation and chromatographic separation of the cytoplasmic content, a peptidoglycan [14C]UDP pentapeptide containing lanthionine was isolated. This finding indicates that lanthionine is synthesized and incorporated as such during the assembly of the peptidoglycan.
Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families
Authors: Munson M A , Baumann P , Clark M A , Baumann L , Moran N A , Voegtlin D J , Campbell B C
Published: November 2016

Aphids (superfamily Aphidoidea) contain eubacterial endosymbionts localized within specialized cells (mycetocytes). The endosymbionts are essential for the survival of the aphid hosts. Sequence analyses of the 16S rRNAs from endosymbionts of 11 aphid species from seven tribes and four families have indicated that the endosymbionts are monophyletic. Furthermore, phylogenetic relationships within the symbiont clade parallel the relationships of the corresponding aphid hosts. Our findings suggest that this endocytobiotic association was established in a common ancestor of the four aphid families with subsequent diversification into the present species of aphids and their endosymbionts.
Specific DNA binding of the TraM protein to the oriT region of plasmid R100
Authors: Abo T , Inamoto S , Ohtsubo E
Published: November 2016

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.
Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli
Authors: Eggen R I , Geerling A C , Boshoven A B , de Vos W M
Published: November 2016

In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs). The acs gene, coding for the single Acs subunit, was isolated from a genomic library of M. soehngenii DNA in Escherichia coli by using antiserum raised against the purified Acs. After introduction in E. coli, the acs gene was expressed, resulting in the production of an immunoreactive protein of 68 kDa, which is approximately 5 kDa smaller than the known size of purified Acs. In spite of this difference in size, the Acs enzymes are produced in similar quantities in E. coli and M. soehngenii and show comparable specific activities. Upstream from the acs gene, consensus archaeal expression signals were identified. Immediately downstream from the acs gene there was a putative transcriptional stop signal. The amino acid sequence deduced from the nucleotide sequence of the acs gene showed homology with those of functionally related proteins, i.e., proteins involved in the binding of coenzyme A, ATP, or both.
Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression
Authors: Lehnherr H , Guidolin A , Arber W
Published: November 2016

Amber mutants of bacteriophage P1 were used to identify functions involved in late regulation of the P1 lytic growth cycle. A single function has been genetically identified to be involved in activation of the phage-specific late promoter sequence Ps. In vivo, P1 gene 10 amber mutants fail to trans activate a lacZ operon fusion under the transcriptional control of promoter Ps. Several P1 segments, mapping around position 95 on the P1 chromosome, were cloned into multicopy plasmid vectors. Some of the cloned DNA segments had a deleterious effect on host cells unless they were propagated in a P1 lysogenic background. By deletion and sequence analysis, the harmful effect could be delimited to a 869-bp P1 fragment, containing a 453-bp open reading frame. This open reading frame was shown to be gene 10 by sequencing the amber mutation am10.1 and by marker rescue experiments with a number of other gene 10 amber mutants. Gene 10 codes for an 18.1-kDa protein showing an unusually high density of charged amino acid residues. No significant homology to sequences present in the EMBL/GenBank data base was found, and the protein contained none of the currently known DNA-binding motifs. An in vivo trans activation assay system, consisting of gene 10 under the transcriptional control of an inducible promoter and a gene S/lacZ fusion transcribed from Ps, was used to show that gene 10 is the only phage-encoded function required for late promoter activation.
Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus
Authors: Hinnebusch J , Barbour A G
Published: November 2016

Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.
Growth stage signal transduction and the requirements for srfA induction in development of competence
Authors: Hahn J , Dubnau D
Published: November 2016

srfA is an operon needed for the development of genetic competence in Bacillus subtilis. This operon is normally expressed at a low level during growth, and its transcription increases sharply just before the transition to stationary phase. The genetic requirements for the full expression of srfA were previously examined in several laboratories and shown to include spo0A, spo0H, spo0K, comQ, and comA. In the present study these results were confirmed with an isogenic set of strains. We have also shown that comP is needed for srfA expression but that other regulatory genes required for competence (degU, sin, and abrB) are not needed for the expression of srfA. We have used the expression of srfA under control of the regulatable Pspac promoter to study the kinetics of competence development and to determine whether the genes ordinarily required for expression of srfA are needed for any additional roles during the development of competence. When expression of srfA was driven from Pspac, competence was expressed constitutively throughout growth. Furthermore, when srfA was expressed from Pspac, the spo0K, comQ, comP, and comA determinants were no longer required for the expression of competence. We conclude therefore that the multiple signals which trigger the initiation of competence development in relation to growth stage are ordinarily received prior to the increase in srfA expression. We propose that these signals are mediated by the products of spo0K, comQ, comP, and comA, resulting in the phosphorylation of ComA by ComP. This in turn would enable ComA to function as a positive transcription factor for srfA, leading to the elaboration of the srfA product(s) and the consequent initiation of competence. We also propose that this is the major, and possibly the only, role for the spo0K, comQ, comP, and comA products during competence development.
Evidence for two sets of structural genes coding for ribulose bisphosphate carboxylase in Thiobacillus ferrooxidans
Authors: Kusano T , Takeshima T , Inoue C , Sugawara K
Published: November 2016

Previously, we reported the cloning of the ribulose-1,5-bisphosphate carboxylase genes (rbcL1-rbcS1) of Thiobacillus ferrooxidans Fe1 (T. Kusano, K. Sugawara, C. Inoue, and N. Suzuki, Curr. Microbiol. 22:35-41, 1991). With these genes as probes, a second set of ribulose-1,5-bisphosphate carboxylase genes (rbcL2-rbcS2) was identified in the same strain and cloned. rbcL1 and rbcL2 encode the large subunits, and rbcS1 and rbcS2 encode the small subunits. Similar restriction patterns between these gene sets suggested a high level of sequence homology. In fact, sequence analysis showed that a 2.2-kb region, including the entire large and small subunit structural genes, was totally conserved in rbcL1-rbcS1 and rbcL2-rbcS2. The rbcL1 (rbcL2) and rbcS1 (rbcS2) genes were 1,422 and 333 bp in length and encoded 473- and 110-amino-acid proteins, respectively. The genes were separated by a 90-bp spacer sequence and were preceded by possible ribosome-binding sites. The N-terminal amino acid sequences of the subunit proteins, synthesized in Escherichia coli, were determined by Edman degradation and found to agree with the deduced amino acid sequences, except for the N-terminal methionine residue. The transcriptional start site of the rbc genes was determined by primer extension, and the size of the rbc transcript was estimated to be about 2.1 kb, suggestive of the cotranscription of rbcL1-rbcS1 and/or rbcL2-rbcS2 mRNAs. Comparisons of amino acid sequences of both subunits with those of other organisms revealed that the ribulose-1,5-bisphosphate carboxylase of T. ferrooxidans, a chemoautotrophic bacterium, is phylogenetically closer to the photosynthetic bacterium Chromatium vinosum than to another chemoautotrophic bacterium, Alcaligenes eutrophus.
A survey of the heat shock response in four Streptomyces species reveals two groEL-like genes and three groEL-like proteins in Streptomyces albus
Authors: Guglielmi G , Mazodier P , Thompson C J , Davies J
Published: November 2016

A survey of the heat shock response was carried out in a series of streptomycetes. Four major heat shock proteins (HSPs) were observed in each of four species (Streptomyces albus, S. lividans, S. parvulus, S. viridochromogenes) after pulse labeling with [35S]methionine and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three corresponded to the major procaryotic HSPs Lon, DnaK, and GroEL on the basis of their apparent molecular masses (94 to 100, 70, and 56 to 58 kDa, respectively). In addition, a smaller protein (16 to 18 kDa) was detected in all species but was most dramatically induced in S. albus. Consequently, studies focused on this species. As in other procaryotic systems, thermal induction (elicited by a shift from 30 degrees C to 41 degrees C) of the 70- and 94-kDa proteins was transient and expression returned to uninduced levels after 60 min. In contrast, the 56- to 58-kDa (GroEL) and 18-kDa proteins (HSP18) remained induced for more than 2 h. Two-dimensional gel electrophoresis allowed resolution of at least eight S. albus HSPs. HSP56-58 was composed of multiple acidic protein species, whereas HSP18 appeared to be basic. In spite of these differences in their physical characteristics, the N-terminal peptide sequence of HSP18 was similar to those of GroEL-like proteins found in other organisms and identical to one of the HSP56-58 species. In fact, N-terminal amino acid analysis of the S. albus 56- to 58-kDa species showed that it was composed of two proteins that differed in 3 of 10 positions, an observation that was supported by the detection of two groEL-like genes by Southern hybridization. The amino acid sequence of one of these proteins was identical to that of HSP18. Pulse-chase experiments did not reveal evidence of posttranslational processing of either HSP56-58 or HSP18.
Bacillus subtilis CheN, a homolog of CheA, the central regulator of chemotaxis in Escherichia coli
Authors: Fuhrer D K , Ordal G W
Published: November 2016

The Bacillus subtilis cheN gene was isolated, sequenced, and expressed. It encodes a large negatively charged protein with a molecular weight of approximately 74,000. The predicted protein sequence has 33 to 34% identity with the Escherichia coli and Salmonella typhimurium CheA and Myxococcus xanthus FrzE sequences. These proteins are found to autophosphorylate and are members of the same histidine kinase signal modulating family. CheN has several conserved regions (including the histidine that is phosphorylated in CheA) that coincide with other autophosphorylated signal transducers. A null mutant is defective in attractant-induced methanol formation and shows no behavioral response to chemoeffectors. These results imply that in B. subtilis the mechanism of chemotaxis involves phosphoryl transfer similar to that in E. coli. However, the CheN null mutant mostly tumbles, whereas CheA mutants swim smoothly, and only in B. subtilis does excitation lead to methyl transfer and methanol formation. Thus, the overall mechanism of chemotaxis is different in the two organisms.
Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene
Authors: Dattananda C S , Rajkumari K , Gowrishankar J
Published: November 2016

Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.
Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium
Authors: Lombardo M J , Bagga D , Miller C G
Published: November 2016

oxrB8, a mutation that diminishes the anaerobic induction of pepT and other anaerobically regulated, oxrA (fnr)-dependent Salmonella typhimurium genes, is an allele of rpoA, the gene for the alpha subunit of RNA polymerase. Four additional rpoA mutations that affect anaerobic pepT expression have been isolated after localized mutagenesis of the rpoA region. All but one of these rpoA mutations appear to have relatively specific effects on genes that require the OxrA (FNR) protein, a positive transcriptional regulator of a family of anaerobically expressed genes. All of these mutations lead to amino acid substitutions in the C-terminal region of the alpha subunit. These results taken with a number of previous observations suggest a role for the alpha subunit in the interaction between RNA polymerase and positive transcriptional regulatory proteins. They also suggest that the C-terminal region of alpha is important for these interactions.
Role of the pheromone-inducible surface protein Asc10 in mating aggregate formation and conjugal transfer of the Enterococcus faecalis plasmid pCF10
Authors: Olmsted S B , Kao S M , van Putte L J , Gallo J C , Dunny G M
Published: November 2016

The high transfer frequency of pheromone-inducible conjugative plasmids of Enterococcus faecalis in liquid culture is due in part to the formation of mating aggregates. These aggregates result from the interaction of two surface components, aggregation substance (AS), which is plasmid encoded, and the chromosomally encoded binding substance (BS). In the accompanying paper (S.-M. Kao, S. B. Olmsted, A. S. Viksnins, J.C. Gallo, G. M. Dunny, J. Bacteriol, 173:7650-7664, 1991), the sequence of the prgB gene encoding the AS molecule (Asc10) produced by pheromone-induced cells carrying plasmid pCF10 is presented. Here we report the results of genetic and immunological experiments which define the role of Asc10 in aggregation and plasmid transfer. These data indicate expression of AS on the surface of an E. faecalis cell and its binding to BS expressed on a second cell are required for the formation of a mating pair and the efficient transfer of pCF10 in liquid matings. However, the orientation of the receptors was not critical for transfer; ie., AS expressed on recipient cells could facilitate plasmid transfer via binding to BS on the donor. Our results suggest that additional (as yet unidentified) products are involved in forming the channel that ultimately serves to transfer the DNA, with AS-BS binding serving primarily to generate the initial attachment between cells. The putative prgC gene product, identified by DNA sequencing (data presented in the accompanying paper), could be involved in transfer events occurring subsequent to aggregation.
Deletion analysis of the F plasmid oriT locus
Authors: Fu Y H , Tsai M M , Luo Y N , Deonier R C
Published: November 2016

Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis. DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans. Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval. Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site. Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function. These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.
Induction and substrate specificity of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae Y222
Authors: Wright G D , Honek J F
Published: November 2016

The potential inducibility of the lanosterol 14 alpha-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37 degrees C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[a]pyrene.
Transposition of Tn5096 and other IS493 derivatives in Streptomyces griseofuscus
Authors: Solenberg P J , Baltz R H
Published: November 2016

Tn5096 was constructed by inserting an apramycin resistance gene, aac(3)IV, into IS493 from Streptomyces lividans. By using conventional and pulsed-field gel electrophoresis, Tn5096 and related transposons were shown to insert into many different locations in the Streptomyces griseofuscus chromosome and in two linear plasmids. On insertion into the target site CANTg, 3 bp appeared to be duplicated. Independent transpositions were obtained by delivery of the transposon from a temperature-sensitive plasmid. The frequency of auxotrophy among cultures containing transpositions was about 0.2%.
Transcriptional analysis, translational analysis, and sequence of the kilA-tellurite resistance region of plasmid RK2Ter
Authors: Walter E G , Thomas C M , Ibbotson J P , Taylor D E
Published: November 2016

The tellurite resistance (Ter) determinant of the IncP alpha plasmid RK2Ter, a variant of RK2 (also called RP4), is located between the kilA and korA genes involved in plasmid replication control. Transcriptional and translational fusions were constructed between the gene for beta-galactosidase and the kilA and Ter genes by using the transpositional phage mini-Mu. These fusions indicated that the Ter genes are transcribed in the same direction as kilA and that transcription and translation of the cloned kilA gene are occurring and may not be lethal to the bacterial cell even in the absence of korA. The nucleotide sequence of this region was determined, and three open reading frames (ORFs) were identified. The first ORF codes for KilA, a 28-kDa hydrophilic protein. The second ORF, telA, codes for a hydrophilic protein of 42 kDa. The third ORF, telB, codes for a hydrophobic protein of 32 kDa. This protein appears to be located in the inner membrane of the bacterial cell, since fusions of TelB to alkaline phosphatase were obtained by using TnphoA. All three proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after overproduction using the T7 RNA polymerase/promoter system. The same three proteins were produced when Tes and Ter derivatives of RP4 were expressed in an in vitro transcription-translation system. A single Ser-to-Cys missense mutation in telB was found to be responsible for mutation of RK2 to Ter.
Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein
Authors: Dellis S , Filutowicz M
Published: November 2016

Integration host factor (IHF) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of Escherichia coli plasmid R6K. We examined the ability of R6K origins to replicate in cells lacking either of the two subunits of IHF. As shown previously, the gamma origin cannot replicate in IHF-deficient cells. However, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of R6K or production of normal levels of mutant pi proteins which exhibit relaxed replication control. The copy number of plasmids containing the primary R6K origins (alpha and beta) is substantially reduced in IHF-deficient bacteria. Furthermore, replication of these plasmids is completely inhibited if the IHF-deficient strains contain a helper plasmid producing additional wild-type pi protein. IHF protein has previously been shown to bind to two sites within the gamma origin. These sites flank a central repeat segment which binds pi protein. We propose a model in which IHF binding to its sites reduces the replication inhibitor activity of pi protein at all three R6K origins.
Transfer of the genes for the StyLTI restriction-modification system of Salmonella typhimurium to strains lacking modification ability results in death of the recipient cells and degradation of their DNA
Authors: De Backer O , Colson C
Published: November 2016

The genes encoding the restriction-modification system StyLTI of Salmonella typhimurium were inserted in vivo into the conjugative plasmid pULB21. This allowed us to transfer the StyLTI genes at a very high frequency and to monitor the fate of recipient cells after mating. Transfer of the StyLTI restriction and modification genes into a modificationless recipient was lethal and resulted in degradation of the cell's DNA. This indicates that, in contrast to any other known restriction-modification systems, StyLTI cannot be established after horizontal transfer into a naive host.
Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775
Authors: Singer J T , Schmidt K A , Reno P W
Published: November 2016

Insertions were created in three iron uptake genes in plasmid pJM1 of Vibrio anguillarum 775 to assess their in vivo effects on virulence in fish. Insertions that blocked p40, pOM2, and pAngR expression resulted in iron uptake-negative strains and in 4.2 x 10(5)-, 8.8 x 10(5)-, and 2.5 x 10(5)-fold attenuations in virulence, respectively. A strain with an insertion in the pAngR coding region still synthesized significant constitutive levels of the outer membrane protein pOM2 and persisted in fish for at least 14 days postinjection. The results demonstrate a direct relationship between virulence and three pJM1-encoded gene products and also the feasibility of constructing live attenuated strains of V. anguillarum that might be useful in future vaccines.
Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella
Authors: Kostrzynska M , Betts J D , Austin J W , Trust T J
Published: November 2016

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.
Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro
Authors: Lünsdorf H , Niedrig M , Fiebig K
Published: November 2016

The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.
RNase T affects Escherichia coli growth and recovery from metabolic stress
Authors: Padmanabha K P , Deutscher M P
Published: November 2016

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.