bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism
Authors: Ernst A , Black T , Cai Y , Panoff J M , Tiwari D N , Wolk C P
Published: November 2016

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.
Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS-DegU signalling system
Authors: Crutz A M , Steinmetz M
Published: November 2016

The adjacent sacX and sacY genes are involved in sucrose induction of the Bacillus subtilis sacB gene by an antitermination mechanism. sacB, encoding the exoenzyme levansucrase, is also subject to regulation by the DegS-DegU signalling system. Using sacXY'-lacZ and sacX'-lacZ fusions, we show that the transcription of the sacX and sacY genes is both inducible by sucrose and regulated by DegU. sacX and sacY appear to constitute an operon, since the deletion of the sacX leader region abolished the expression of a sacXY'-lacZ fusion. The degU-dependent promoter was located by deletion analysis and reverse transcriptase mapping 300 nucleotides upstream from the sacX initiator codon. Sucrose induction of the sacX'-lacZ fusion requires either SacY or the homologous SacT antiterminator, which is involved in sucrose induction of the intracellular sucrase gene (sacPA operon). Sequence analysis of the sacX leader region revealed (20 nucleotides downstream from the transcription start site) a putative binding site for these regulators; however, no structure resembling a rho-independent terminator could be found overlapping this site, unlike the situation for sacPA and sacB. Deletion of a segment of the leader region located 100 nucleotides downstream from this site led to constitutive expression of the sacXY'-lacZ and sacX'-lacZ fusions. These results suggest that the mechanism of sucrose induction of sacXY is different from that of sacPA and sacB.
The NodD protein does not bind to the promoters of inducible nodulation genes in extracts of bacteroids of Rhizobium leguminosarum biovar viciae
Authors: Schlaman H R , Lugtenberg B J , Okker R J
Published: November 2016

In a previous study, we showed that in bacteroids, transcription of the inducible nod genes does not occur and expression of nodD is decreased by 65% (H. R. M. Schlaman, B. Horvath, E. Vijgenboom, R.J.H. Okker, and B. J. J. Lugtenberg, J. Bacteriol. 173:4277-4287, 1991). In the present study, we show, using gel retardation, that in crude extracts of bacteroids of Rhizobium leguminosarum biovar (bv.) viciae, NodD protein does not bind to the nodF, nodM, and nodO box and that it binds only weakly to the nodA box. Binding of NodD from bacteroids to nod box DNA could be restored by mild proteinase K treatment, indicating that NodD is present in bacteroids in an altered form or complex which prevents its binding to nod box DNA. In addition, a novel nodA box DNA-protein complex was found which is specific for the nodA promoter region. This novel complex was formed neither with material from cultured bacterial cells nor with an extract from uninfected roots, and it did not contain NodD but another protein. These results are consistent with the hypothesis that the protein present in the novel retardation complex acts as a transcriptional repressor causing the decreased nodD expression in bacteroids. Such a repressor also explains the lack of nodABCIJ transcription despite the weak NodD binding to the nodA box.
Structural characterization and corepressor binding of the Escherichia coli purine repressor
Authors: Choi K Y , Zalkin H
Published: November 2016

The Escherichia coli purine repressor, PurR, binds to a 16-bp operator sequence and coregulates the genes for de novo synthesis of purine and pyrimidine nucleotides, formation of a one-carbon unit for biosynthesis, and deamination of cytosine. We have characterized the purified repressor. Chemical cross-linking indicates that PurR is dimeric. Each subunit has an N-terminal domain of 52 amino acids for DNA binding and a C-terminal 289-residue domain for corepressor binding. Each domain was isolated after cleavage by trypsin. Sites for dimer formation are present within the corepressor binding domain. The corepressors hypoxanthine and guanine bind cooperatively to distinct sites in each subunit. Competition experiments indicate that binding of one purine abolishes cooperativity and decreases the affinity and the binding of the second corepressor. Binding of each corepressor results in a conformation change in the corepressor binding domain that was detected by intrinsic fluorescence of three tryptophan residues. These experiments characterize PurR as a complex allosteric regulatory protein.
New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity
Authors: Larminat F , Cazaux C , Germanier M , Defais M
Published: November 2016

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.
Decreased function of the class B tetracycline efflux protein Tet with mutations at aspartate 15, a putative intramembrane residue
Authors: McMurry L M , Stephan M , Levy S B
Published: November 2016

The aspartate 15 residue within the first predicted intramembrane helix of the tetracycline efflux protein Tet has been conserved in four tetracycline resistance determinants from gram-negative bacteria. Its replacement in class B Tet by tyrosine, histidine, or asparagine resulted in a 60 to 85% loss of tetracycline resistance and a similar loss of tetracycline-proton antiport. The tyrosine and histidine substitutions lowered the Vmax of the efflux system by some 90% but did not alter the Km. The asparagine substitution raised the Km over 13-fold, while the Vmax was equal to or greater than that of the wild type. Therefore, although the nature of its role is unclear, aspartate 15 is important for normal Tet function.
Analysis of a peptidoglycan hydrolase gene from Staphylococcus aureus NCTC 8325
Authors: Wang X , Mani N , Pattee P A , Wilkinson B J , Jayaswal R K
Published: November 2016

We have investigated the expression of the peptidoglycan hydrolase gene (lytA) of Staphylococcus aureus NCTC 8325. Results from in vitro transcription-translation analysis, maxicell experiments, and Northern (RNA) blot analysis suggest that the lytA gene encodes a polypeptide of M(r) approximately 50,000. Physical mapping data indicate that the lytA gene originated from prophage 11 in the NCTC 8325 strain.
Size and stability of the genomes of the myxobacteria Stigmatella aurantiaca and Stigmatella erecta
Authors: Neumann B , Pospiech A , Schairer H U
Published: November 2016

Genomic DNA of Stigmatella aurantiaca DW 4/3.1 was restricted with the rare-cutting endonucleases AseI and SpeI. The restriction pattern derived is composed of 33 AseI and 25 SpeI fragments, whose total size amounts to approximately 9,350 kbp. Genomic fingerprint analysis of chromosomal DNA from several S. aurantiaca isolates further revealed five completely different SpeI and AseI fingerprints and one distinct fingerprint for Stigmatella erecta. In addition, minor variations between the genome sizes of these isolates were observed.
DNA polymerase I modulates inducible stable DNA replication in Escherichia coli
Authors: Ruscitti T , Linn S
Published: November 2016

Mutants of Escherichia coli lacking RNase HI activity and cells induced for the SOS response express modes of DNA replication independent of protein synthesis, called constitutive and induced stable DNA replication, respectively. We report here that mutants deleted for the polA gene express induced stable DNA replication at approximately 25-fold the rate of wild-type cells, whereas constitutive stable DNA replication is not enhanced.
Inhibition of cell division initiation by an imbalance in the ratio of FtsA to FtsZ
Authors: Dewar S J , Begg K J , Donachie W D
Published: November 2016

Elevated levels of FtsA protein block cell division at a very early stage, similar to that caused by inhibition of the action of FtsZ. In contrast, overexpression of FtsA and FtsZ together does not block division. A specific ratio of FtsA to FtsZ protein, therefore, is required for cell division.
Characterization of the gill symbiont of Thyasira flexuosa (Thyasiridae: Bivalvia) by use of polymerase chain reaction and 16S rRNA sequence analysis
Authors: Distel D L , Wood A P
Published: November 2016

Comparative molecular sequence (16S rRNA) analysis methods were used to identify and characterize the symbionts of Thyasira flexuosa independently of pure culture techniques and to compare these symbionts with the previously reported putative symbiont isolate, Thiobacillus thyasiris TG-2 (A. P. Wood and D. P. Kelly, Arch. Microbiol. 152:160-166, 1989). Polymerase chain reaction amplification using 16S rRNA primers specific for eubacteria was used to amplify a single unique sequence from the gill tissue of T. flexuosa. This sequence is phylogenetically most closely related to the 16S rRNA genes of known symbionts of lucinid clams and is distinct from those determined for strain TG-2 and other known bacteria. Strain TG-2 most closely resembles a free-living, chemolithoautotrophic bacterium known to be associated with the surfaces of thiotrophic bivalve shells, suggesting that this strain is a contaminant and not the authentic intracellular symbiont of T. flexuosa.
Cloning, sequencing, and expression of the pantothenate kinase (coaA) gene of Escherichia coli
Authors: Song W J , Jackowski S
Published: November 2016

Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. The structural gene (coaA) located at 90 min of the Escherichia coli chromosome was cloned and sequenced. The coaA gene was transcribed in the opposite direction to the flanking genes birA and thrU and produced a single 1.1-kb transcript. Translation of the coaA gene produced two protein products (36.4 and 35.4 kDa) that differed by eight amino acids at the amino terminus. The poor homology of the coaA promoter region to consensus E. coli promoter sequences and the low frequency of optimal codon usage (0.565) were consistent with the low abundance of pantothenate kinase. Strains containing multiple copies of the coaA gene possessed 76-fold-higher specific activity of pantothenate kinase; however, there was only a 2.7-fold increase in the steady-state level of CoA. These data corroborate the conclusion that regulation of pantothenate kinase activity by feedback inhibition is the critical factor controlling the intracellular CoA concentration.
Coordination of expression of Zymomonas mobilis glycolytic and fermentative enzymes: a simple hypothesis based on mRNA stability
Authors: Mejia J P , Burnett M E , An H , Barnell W O , Keshav K F , Conway T , Ingram L O
Published: November 2016

Although Zymomonas mobilis is prototrophic, glycolytic and fermentative enzymes (ethanologenic enzymes) constitute over half of the cytoplasmic protein. In this study, transcript stability, functional message pools, and the abundance of cytoplasmic products were compared for genes encoding eight of these essential enzymes. The transcripts of all were very stable, with half-lives ranging from 8 to 18 min. This transcript stability is proposed as an important feature in Z. mobilis that may distinguish highly expressed genes for energy generation from biosynthetic genes, which are required at much lower levels. The evolution of multiple promoters to enhance transcription from single-copy genes, of structural features that alter translational efficiency, and of differences in protein turnover is hypothesized to serve a subordinate role in the regulation of Z. mobilis gene expression. Among the eight ethanologenic genes examined, differences in transcript stability were found to directly correlate with differences in functional message pools and cytoplasmic protein levels. These differences in transcript stability are hypothesized to have evolved as a primary mechanism to balance the levels of individual enzymes within the glycolytic and fermentative pathways.
Detection of growth sites in and protomer pools for the sheath of Methanospirillum hungatei GP1 by use of constituent organosulfur and immunogold labeling
Authors: Southam G , Beveridge T J
Published: November 2016

Methanospirillum hungatei GP1 integrated approximately 9% of cellular [35S]cysteine into its sheath. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels revealed that [35S]cysteine was confined to the proteins released by the sodium dodecyl sulfate-beta-mercaptoethanol-EDTA solubilization method (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) and was not present in the proteins released by treatment with phenol (G. Southam and T. J. Beveridge, J. Bacteriol. 174:935-946, 1992). Limited labeling of exposed sulfhydryl groups on hoops produced from sheath material suggested that most organosulfur groups occur within hoops and therefore help contribute to resilience. Electron microscopic autoradiography demonstrated that sheath growth, which is most active at the sites of cell division (spacer region), occurs through the de novo development of hoops. For growth to occur in the spacer region, sheath precursors must transverse several periodic envelope layers, including the cell wall (a single layer) and the various lamellae of the spacer plug (T. J. Beveridge, G. D. Sprott, and P. Whippey, J. Bacteriol. 173:130-140, 1991).
Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes
Authors: Citti C , Maréchal-Drouard L , Saillard C , Weil J H , Bové J M
Published: November 2016

From the total tRNAs of Spiroplasma citri, we isolated and purified two tRNA(Trp) species by using chromatography on an RPC-5 column followed by denaturing polyacrylamide gel electrophoresis. The sequence of the two tRNAs, as well as the sequences of the corresponding genes, were determined. One of the two tRNA(Trp) species has a CCA anticodon and is able to pair with the universal UGG tryptophan codon, while the second has a U*CA (U* is a modified uridine) anticodon and is able to pair with UGA but also with UGG in accordance with the "U:N wobble" rule. Thus, in S. citri, UGA is not a stop codon but codes for tryptophan. The two tRNA(Trp) genes, together with a third tRNA gene, tRNA(Ser) (CGA), belong to a single transcription unit. The nucleotide sequences of the two tRNA(Trp) species show 82.9% similarity. The two spiroplasmal tRNA(Trp) species can be aminoacylated by using an aminoacyl-tRNA synthetase fraction from S. citri. In contrast, the enzyme fraction from Escherichia coli aminoacylates tRNA(Trp) (CCA) but not tRNA(Trp) (U*CA).
Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP
Authors: Puziss J W , Harvey R J , Bassford P J
Published: November 2016

Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M. N. Hall, J. Gabay, and M. Shwartz, EMBO J. 2:15-19, 1983; S. Inouye, X. Soberon, T. Franceschini, K. Nakamura, K. Itakura, and M. Inouye, Proc. Natl. Acad. Sci. USA 79:3438-3441, 1982; J. W. Puziss, J. D. Fikes, and P. J. Bassford, Jr., J. Bacteriol. 171:2302-2311, 1989). This result has been interpreted as evidence that the hydrophilic segment is part of a mechanism that obligatorily couples translation to protein export. We have investigated the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the export and synthesis of MBP. Deletion of the entire hydrophilic segment from the MBP signal peptide resulted in a defect in MBP export, as well as a dramatic reduction in total MBP synthesis. Suppressor mutations that lie upstream of the malE coding region were isolated. These mutations do not affect MBP export but instead were shown to partially restore MBP synthesis by increasing the efficiency of MBP translational initiation. In addition, analysis of a series of substitution mutations in the second codon of certain malE alleles demonstrated that MBP export and synthesis can be independently affected by mutations in the hydrophilic segment. Finally, analysis of alterations in the hydrophilic segment of the ribose-binding protein signal peptide fused to the mature moiety of the MBP has revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation. Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli.
Tryptophan biosynthesis genes in Lactococcus lactis subsp. lactis
Authors: Bardowski J , Ehrlich S D , Chopin A
Published: November 2016

The Lactococcus lactis chromosomal region containing the seven structural genes required for tryptophan biosynthesis was characterized by cloning and sequencing. All of the trp genes were identified by the homology of their products with known Trp proteins from other organisms. The identification was confirmed for five genes by their ability to complement trp mutations in Escherichia coli. The seven structural genes are present in the order trpEGDCFBA and span a 7,968-bp segment. Each gene is preceded by a putative ribosome binding site complementary to the 3' end of the L. lactis 16S rRNA. Three pairs of genes (trpG-trpD, trpC-trpF, and trpB-trpA) overlap, and there is intercistronic spacing of 124, 46, and 585 bp between the trpE-trpG, trpD-trpC, and trpF-trpB gene pairs, respectively. No gene fusion was found. Upstream of the trp genes, a 457-bp noncoding DNA segment contains several regions fitting the consensus for gram-positive promoters and one region strongly resembling a transcription terminator. However, it seems unlikely that an attenuation mechanism similar to the one found in E. coli regulates tryptophan biosynthesis in L. lactis, since no potential leader peptide was detected. We propose that a mechanisms resembling that described in Bacillus spp. can regulate trp genes expression in L. lactis.
Role of the origin of transfer in termination of strand transfer during bacterial conjugation
Authors: Bhattacharjee M , Rao X M , Meyer R J
Published: November 2016

Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oriTs, with transfer initiated at one and terminated at the other, the inverted repeat within oriT is important for termination. Deletion of the outer arm reduces the termination frequency; those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved. We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.
Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity
Authors: Waters V L , Strack B , Pansegrau W , Lanka E , Guiney D G
Published: November 2016

Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncP alpha plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.
Saccharomyces cerevisiae has distinct adaptive responses to both hydrogen peroxide and menadione
Authors: Jamieson D J
Published: November 2016

Treatment of Saccharomyces cerevisiae cells with low concentrations of either hydrogen peroxide or menadione (a superoxide-generating agent) induces adaptive responses which protect cells from the lethal effects of subsequent challenge with higher concentrations of these oxidants. Pretreatment with menadione is protective against cell killing by hydrogen peroxide; however, pretreatment with hydrogen peroxide is unable to protect cells from subsequent challenge with menadione. This suggests that the adaptive responses to these two different oxidants may be distinct.
Biosynthetic pathways of the osmolytes N epsilon-acetyl-beta-lysine, beta-glutamine, and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses
Authors: Roberts M F , Lai M C , Gunsalus R P
Published: November 2016

Methanohalophilus strain FDF1 synthesizes beta-glutamine, betaine, and N epsilon-acetyl-beta-lysine as osmoprotective agents when the cells are grown in high external concentrations of NaCl. Nuclear magnetic resonance spectroscopic analyses of 13CH3OH-12CO2 label incorporation by the cells provide information on the biosynthetic pathways of these organic osmolytes. The labeling studies indicate that Methanohalophilus strain FDF1 produces glutamate and beta-glutamine via a partial oxidative Krebs pathway. 13C labeling of betaine is consistent with methylation of glycine generated from serine (via serine hydroxymethyltransferase). The labeling pattern for N epsilon-acetyl-beta-lysine is consistent with the synthesis of its precursor alpha-lysine occurring by the diaminopimelate pathway in these cells.
Identification of Bacillus subtilis genes for septum placement and shape determination
Authors: Levin P A , Margolis P S , Setlow P , Losick R , Sun D
Published: November 2016

The Bacillus subtilis divIVB1 mutation causes aberrant positioning of the septum during cell division, resulting in the formation of small, anucleate cells known as minicells. We report the cloning of the wild-type allele of divIVB1 and show that the mutation lies within a stretch of DNA containing two open reading frames whose predicted products are in part homologous to the products of the Escherichia coli minicell genes minC and minD. Just upstream of minC and minD, and in the same orientation, are three genes whose products are homologous to the products of the E. coli shape-determining genes mreB, mreC, and mreD. The B. subtilis mreB, mreC, and mreD genes are the site of a conditional mutation (rodB1) that causes the production of aberrantly shaped cells under restrictive conditions. Northern (RNA) hybridization experiments and disruption experiments based on the use of integrational plasmids indicate that the mre and min genes constitute a five-cistron operon. The possible involvement of min gene products in the switch from medial to polar placement of the septum during sporulation is discussed.
Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus
Authors: Sakai-Tomita Y , Moritani C , Kanazawa H , Tsuda M , Tsuchiya T
Published: November 2016

Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe.
Characterization of the Bacillus subtilis rpsD regulatory target site
Authors: Grundy F J , Henkin T M
Published: November 2016

The Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, is subject to autogenous regulation. Repression of rpsD expression by excess S4 protein was previously shown to be affected by mutations in the leader region of the gene. A large number of deletion and point mutations in the leader region were generated, and their effect on repression by S4 in vivo was tested. These studies indicated that the required region was within positions +30 to +190 relative to the transcription start point. Replacement of the rpsD promoter with a lac promoter derivative which is expressed in B. subtilis had no effect, indicating that repression by S4 occurs at a level subsequent to transcription initiation. The rpsD leader region was isolated from several Bacillus species. Members of the B. subtilis group, as defined by analysis of 16S rRNA sequence, contained a leader region target site very closely related in structure to that of B. subtilis, despite considerable primary sequence variation; the B. brevis rpsD leader contained some but not all of the structural features found in the regulatory target sites of the other Bacillus species. Very little similarity to the Escherichia coli alpha operon S4 target site was found at either the primary-sequence or the secondary-structure level. Mutagenic and phylogenetic data indicate that the secondary structure of the leader region regulatory target site contains two large stem-loop domains. The first of these helices has a side loop which is essential for autoregulation, is highly conserved among Bacillus rpsD genes, and is similar to a region of 16S rRNA important in S4 binding.
Effects of amino acid substitutions in the -10 binding region of sigma E from Bacillus subtilis
Authors: Jones C H , Tatti K M , Moran C P
Published: November 2016

The sigma subunit of bacterial RNA polymerase is required for specific binding to promoters. One region in most sigma factors makes sequence-specific contacts at the -10 region of its cognate promoters. To test the role of the amino acids in this -10 binding region, we examined the effects of 49 single-amino-acid substitutions in sigma E from Bacillus subtilis. We assayed the effect of each amino acid substitution on spore formation because sigma E is essential for endospore formation in B. subtilis. Our results showed that substitutions at several positions, including the highly conserved aromatic amino acid at position 102, had little or no detectable effect. Substitutions at another position, position 117, produced dominant negative mutations; we suggest that these mutations allow RNA polymerase containing the mutant sigma factor to bind specifically to promoters but prevent transcription initiation. Of the recessive defective alleles, those that produced substitutions at positions 113, 115, and 120 produced the most defective sigma factors. These results suggest that the residues at or near these positions in wild-type sigma E play important roles in sigma E function.
Chemolithoautotrophic assimilation of dinitrogen by Streptomyces thermoautotrophicus UBT1: identification of an unusual N2-fixing system
Authors: Gadkari D , Mörsdorf G , Meyer O
Published: November 2016

Streptomyces thermoautotrophicus UBT1, which was isolated previously from a burning charcoal pile, was shown to utilize N2 as a sole nitrogen source when growing chemolithoautotrophically with CO or H2 plus CO2 under aerobic conditions at 65 degrees C. Doubling times under diazotrophic conditions were 10 h. S. thermoautotrophicus is a new CO- or H2-oxidizing, obligately chemolithoautotrophic, thermophilic, free-living, aerobic, N2-fixing streptomycete. Its ability to fix N2 was also evident from (i) the incorporation of substantial amounts of 15N2 (about 13%) into cell material, (ii) the formation of H2 during diazotrophic growth, (iii) the repression of 15N2 assimilation and H2 formation by ammonia, and (iv) culture growth yields with N2 as a nitrogen source that were significantly higher than those without any added nitrogen compounds (ca. 2.4 versus < 0.1 mg [dry weight]). The N2-fixing system of S. thermoautotrophicus exhibited several properties not apparent in the diazotrophic bacteria studied so far, since it was (i) incapable of reducing acetylene to ethylene or ethane and (ii) resistant to inhibition by acetylene or ethylene (5% [vol/vol] each), CO (40 to 70% [vol/vol]), or H2 (40% [vol/vol]). Under stringent conditions, nifH and nifDK gene probes from Klebsiella pneumoniae did not hybridize with total DNA from S. thermoautotrophicus.
Function of the N-terminal half of RepA in activation of Rts1 ori
Authors: Terawaki Y , Itoh Y , Zeng H , Hayashi T , Tabuchi A
Published: November 2016

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.
Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli
Authors: Wu Y , Patil R V , Datta P
Published: November 2016

Anaerobic expression of the tdcABC operon of Escherichia coli requires cyclic AMP and the catabolite gene activator protein (CAP). Purified CAP binds to a 30-bp sequence in the tdc promoter between positions -55 and -26, and a mutant CAP site with base substitutions at positions -48, -47, and -45 failed to bind CAP and also drastically reduced the beta-galactosidase expression from a tdcB'-'lacZ fusion plasmid. Recently, we showed that efficient expression of the tdc operon also requires a functional integration host factor (IHF) and an IHF-binding site in the tdc promoter between positions -118 and -88. The levels of beta-galactosidase activity from the tdcB'-'lacZ fusion plasmids were also reduced in an IHF-deficient strain with the wild-type or mutant plasmid CAP sequence. In vitro footprinting experiments revealed that CAP and IHF occupy their specific binding sites on tdc DNA when they are present separately or together. These regulatory proteins also induced significant bending of the tdc promoter DNA. Our results suggest that CAP and IHF act in concert as positive transcription factors for tdc operon expression in vivo.
Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity
Authors: Nohmi T , Yamada M , Watanabe M , Murayama S Y , Sofuni T
Published: November 2016

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed.
Molecular cloning, sequencing, and overexpression of the structural gene encoding the delta subunit of Escherichia coli DNA polymerase III holoenzyme
Authors: Carter J R , Franden M A , Aebersold R , McHenry C S
Published: November 2016

Using an oligonucleotide hybridization probe, we have mapped the structural gene for the delta subunit of Escherichia coli DNA polymerase III holoenzyme to 14.6 centisomes of the chromosome. This gene, designated holA, was cloned and sequenced. The sequence of holA matches precisely four amino acid sequences obtained for the amino terminus of delta and three internal tryptic peptides. A holA-overproducing plasmid that directs the expression of delta up to 4% of the soluble protein was constructed. Sequence analysis of holA revealed a 1,029-bp open reading frame that encodes a protein with a predicted molecular mass of 38,703 Da. holA may reside downstream of rlpB in an operon, perhaps representing yet another link between structural genes for the DNA polymerase III holoenzyme and proteins involved in membrane biogenesis. These and other features are discussed in terms of genetic regulation of delta-subunit synthesis.
The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations
Authors: Haugan K , Karunakaran P , Blatny J M , Valla S
Published: November 2016

The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.
Altered-function mutations of the transcriptional regulatory gene virG of Agrobacterium tumefaciens
Authors: Han D C , Chen C Y , Chen Y F , Winans S C
Published: November 2016

Three point mutations were isolated in the Agrobacterium tumefaciens virG gene by screening for vir gene expression in the absence of added phenolic inducing compounds. All three mutations were localized in the predicted amino-terminal phosphoryl receiver domain of the protein. One mutant (N54D) bypasses the requirement for VirA and phenolic inducers both for transcriptional activation of all tested vir promoters and for plant tumorigenesis. This mutant also activates vir gene expression efficiently at neutral pH, indicating that the step in induction that is normally stimulated by acid pH occurs before or during VirG phosphorylation. The other two mutants (M13T and H15R) require VirA for activity but are sensitized to low levels of inducing stimuli.
Regulation of the Staphylococcus aureus plasmid pI258 mercury resistance operon
Authors: Chu L , Mukhopadhyay D , Yu H , Kim K S , Misra T K
Published: November 2016

Experiments involving fusion between the Staphylococcus aureus plasmid pI258-encoded mer operon and the reporter gene beta-lactamase, mutational analysis, and trans-complementation studies have shown that the merR gene of pI258, which shows DNA sequence similarity with known merR genes from other bacteria, regulates the expression of the mer operon in vivo. The merR gene product is a trans-acting protein that activates mer operon transcription in the presence of the inducers Hg2+ and Cd2+. A glutathione-S-transferase-MerR fusion protein specifically bound and protected a 27-nucleotide operator sequence from DNase I digestion. This operator sequence is highly homologous with mer operator sequences of other known systems.
Influence of carbon source on cell surface topology of Thermomonospora curvata
Authors: Hostalka F , Moultrie A , Stutzenberger F
Published: November 2016

The appearance of cell surface protuberances in Thermomonospora curvata correlated with cell-bound exoenzymes which could be removed by brief sonication. Mycelia grown on cellulose or xylan had numerous protuberances and retained 20 to 25% of endoglucanase and endoxylanase at cell surfaces, while those grown on pectin or starch had few protuberances and negligible bound pectinase or amylase.
Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins
Authors: Foster P L , Marinus M G
Published: November 2016

In Escherichia coli, epsilon, the proofreading subunit of DNA polymerase III, is encoded by dnaQ. A random search for mutants that affect the expression of dnaQ revealed that mutations in the genes encoding the heat shock proteins (HSPs) DnaK, DnaJ, and GrpE result in dramatic decreases in the cellular levels of epsilon. dnaQ is arranged in an overlapping divergent transcriptional unit with rnhA, which encodes RNase H1, and mutations in the same HSPs also reduced the apparent levels of RNase H1. The HSPs had only small effects on transcriptional fusions to these genes; thus, it is likely that they operate primarily at the protein level. Since survival and mutagenesis after DNA damage are affected by epsilon and RNase H1, HSPs may have a broad influence on various aspects of DNA replication and repair.
Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli
Authors: Yamanaka K , Ogura T , Niki H , Hiraga S
Published: November 2016

The mukB gene encodes a protein involved in chromosome partitioning in Escherichia coli. To study the function of this protein, we isolated from the temperature-sensitive mukB null mutant and characterized 56 suppressor mutants which could grow at 42 degrees C. Ten of the mutants also showed cold-sensitive growth at 22 degrees C. Using one of the cold-sensitive mutants as host, the wild type of the suppressor gene was cloned. The cloned suppressor gene complemented all of the 56 suppressor mutations. DNA sequencing revealed the presence of an open reading frame of 723 bp which could encode a protein of 25,953 Da. The gene product was indeed detected. The previously undiscovered gene, named smbA (suppressor of mukB), is located at 4 min on the E. coli chromosome, between the tsf and frr genes. The smbA gene is essential for cell proliferation in the range from 22 to 42 degrees C. Cells which lacked the SmbA protein ceased macromolecular synthesis. The smbA mutants are sensitive to a detergent, sodium dodecyl sulfate, and they show a novel morphological phenotype under nonpermissive conditions, suggesting a defect in specific membrane sites.
Scattering of the rRNA genes on the physical map of the circular chromosome of Leptospira interrogans serovar icterohaemorrhagiae
Authors: Baril C , Herrmann J L , Richaud C , Margarita D , Girons I S
Published: November 2016

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.
Possible role of Escherichia coli penicillin-binding protein 6 in stabilization of stationary-phase peptidoglycan
Authors: van der Linden M P , de Haan L , Hoyer M A , Keck W
Published: November 2016

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambda pR promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical cells or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6s350). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic overexpression of native PBP6 gave rise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane-bound and the soluble forms of PBP6 were purified to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6s350 formed a 14[C]penicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that of PBP5 and to suggest a role for PBP6 in the stabilization of the peptidoglycan during stationary phase.