bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans
Authors: Bormann C , Möhrle V , Bruntner C
Published: November 2016

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.
The glucose effect and regulation of alpha-amylase synthesis in the hyperthermophilic archaeon Sulfolobus solfataricus
Authors: Haseltine C , Rolfsmeier M , Blum P
Published: November 2016

An alpha-amylase was purified from culture supernatants of Sulfolobus solfataricus 98/2 during growth on starch as the sole carbon and energy source. The enzyme is a homodimer with a subunit mass of 120 kDa. It catalyzes the hydrolysis of starch, dextrin, and alpha-cyclodextrin with similar efficiencies. Addition of exogenous glucose represses production of alpha-amylase, demonstrating that a classical glucose effect is operative in this organism. Synthesis of [35S]-alpha-amylase protein is also subject to the glucose effect. alpha-Amylase is constitutively produced at low levels but can be induced further by starch addition. The absolute levels of alpha-amylase detected in culture supernatants varied greatly with the type of sole carbon source used to support growth. Aspartate was identified as the most repressing sole carbon source for alpha-amylase production, while glutamate was the most derepressing. The pattern of regulation of alpha-amylase production seen in this organism indicates that a catabolite repression-like system is present in a member of the archaea.
Suppression of a signaling defect during Myxococcus xanthus development
Authors: Lee K , Shimkets L J
Published: November 2016

The csgA gene encodes an extracellular protein that is essential for cell-cell communication (C-signaling) during fruiting body development of Myxococcus xanthus. Two transposon insertions in the socABC operon, soc-560 and socC559, restore development to csgA null mutants. Mixing soc-560 csgA cells or socC559 csgA cells with csgA cells at a ratio of 1:1 stimulated the development of csgA cells, suggesting that soc mutations allow cells to produce the C-signal or a similar molecule via a csgA-independent mechanism. The socABC operon contains the following three genes: socA, a member of the short-chain alcohol dehydrogenase gene family; socB, a gene encoding a putative membrane anchoring protein; and socC, a negative autoregulator of socABC operon expression. Both suppressor mutations inactivate socC, leading to a 30- to 100-fold increase in socA transcription; socA expression in suppressor strains is at least 100-fold higher than csgA expression during all stages of development. The amino acid sequence of SocA has 28% identity and 51% similarity with that of CsgA. We suggest that CsgA suppression is due to overproduction of SocA, which can substitute for CsgA. These results raise the possibility that a cell surface dehydrogenase plays a role in C-signaling.
Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis
Authors: Mahairas G G , Sabo P J , Hickey M J , Singh D C , Stover C K
Published: November 2016

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.
Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli
Authors: Koehler D R , Courcelle J , Hanawalt P C
Published: November 2016

We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs.
Phylogenetic analysis of Metabacterium polyspora: clues to the evolutionary origin of daughter cell production in Epulopiscium species, the largest bacteria
Authors: Angert E R , Brooks A E , Pace N R
Published: November 2016

It is rare that there are molecular clues to the evolutionary origin of developmental traits. We have encountered an evolutionary juxtaposition that may explain the origin of the unique replicative morphology of Epulopiscium spp., the largest known bacteria, which reproduce by the internal production of multiple live offspring. We report here a 16S rRNA-based phylogenetic analysis of Metabacterium polyspora, a multiple-endospore-forming, uncultivated inhabitant of guinea pig cecum. Cells of M. polyspora were harvested from cecum contents by sedimentation in a Ficoll gradient and lysed. The bacterial 16S rRNA genes of this lysate were amplified by PCR. Sequence analysis of the cloned PCR products revealed two dominant, closely related 16S rRNA types. In situ hybridization of cecum contents with fluorescently labeled oligonucleotides, diagnostic of these two sequences, showed that they represent distinct strains of M. polyspora. Phylogenetic analyses of the sequences showed that M. polyspora is closely related to Epulopiscium spp. On the basis of this result and other correlations, we propose that the process of sporulation was modified in a predecessor of Epulopiscium spp. to produce live offspring instead of quiescent endospores.
Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required
Authors: Sia E A , Kuehner D M , Figurski D H
Published: November 2016

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events.
Carboxylation of epoxides to beta-keto acids in cell extracts of Xanthobacter strain Py2
Authors: Allen J R , Ensign S A
Published: November 2016

A novel enzymatic reaction involved in the metabolism of aliphatic epoxides by Xanthobacter strain Py2 is described. Cell extracts catalyzed the CO2-dependent carboxylation of propylene oxide (epoxypropane) to form acetoacetate and beta-hydroxybutyrate. The time courses of acetoacetate and beta-hydroxybutyrate formaton indicate that acetoacetate is the primary product of propylene oxide carboxylation and that beta-hydroxybutyrate is a secondary product formed by the reduction of acetoacetate. Analogous C5 carboxylation products were identified with 1,2-epoxybutane as the substrate. In the absence of CO2, propylene oxide and 1,2-epoxybutane were isomerized to form acetone and methyl ethyl ketone, respectively, as dead-end products. The carboxylation of short-chain epoxides to beta-keto acids is proposed to serve as the physiological reaction for the metabolism of aliphatic epoxides in Xanthobacter strain Py2.
Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium
Authors: Enos-Berlage J L , Downs D M
Published: November 2016

purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement. We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6-phosphate [6-P] dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase). We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine.
A deduced Thermomonospora curvata protein containing serine/threonine protein kinase and WD-repeat domains
Authors: Janda L , Tichý P , Spízek J , Petrícek M
Published: November 2016

The gene pkwA coding for a typical WD-repeat protein was found in the chromosome of the bacterium Thermomonospora curvata CCM 3352. Until now WD-repeat proteins were through to be confined to eukaryotes.
Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti
Authors: Holloway P , McCormick W , Watson R J , Chan Y K
Published: November 2016

The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins, The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein presumably forming a copper-processing system. A consensus sequence for an Anr- or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN-binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N2-fixing ability.
A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum
Authors: Preisig O , Zufferey R , Thöny-Meyer L , Appleby C A , Hennecke H
Published: November 2016

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.
Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli
Authors: Liu S Y , Gherardini F C , Matuschek M , Bahl H , Wiegel J
Published: November 2016

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.
Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli
Authors: Brøndsted L , Atlung T
Published: November 2016

The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene have been investigated under different environmental conditions with single-copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase. ArcA and AppY activated transcription of the cyx-appA operon during entry into stationary phase and under anaerobic growth conditions. The expression of the cyx-appA operon was affected by the anaerobic energy metabolism. The presence of the electron acceptors nitrate and fumarate repressed the expression of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high level of expression of the operon was obtained in glucose medium supplemented with formate, in which E. coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented in this paper indicate a clear difference in the regulation of the cyx-appA operon and that of the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function under even more-oxygen-limiting conditions than cytochrome d oxidase. The expression of the appY gene is induced immediately by anaerobiosis, and this anaerobic induction is independent of Fnr, and AppY, but dependent on ArcA. The expression of the appY gene is not affected significantly by the anaerobic energy metabolism, i.e., fermentation versus anaerobic respiration. A model incorporating the anaerobic regulation of the appY gene and the two operons which are controlled by AppY, the hydrogenase 1 (hya) operon and the acid phosphatase (cyx-appA) operon, is presented. The expression of the appY gene is inversely correlated with the growth rate and is induced by phosphate starvation as well as during entry into stationary phase. During oxygen-limiting conditions the stationary-phase induction is partially dependent on ArcA. The alternative sigma factor sigma S has limited influence on the transcription of the appY gene during entry into stationary phase and no effect on the induction by phosphate starvation.
Multiple glucan-binding proteins of Streptococcus sobrinus
Authors: Ma Y , Lassiter M O , Banas J A , Galperín M Y , Taylor K G , Doyle R J
Published: November 2016

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.
Regulatory factors acting at the bacteriophage Mu middle promoter
Authors: Kahmeyer-Gabbe M , Howe M M
Published: November 2016

Lytic development of bacteriophage Mu proceeds through three phases of transcription: early, middle, and late. Initiation of middle transcription from Pm requires the phage-encoded activator, Mor. An examination of the sequences surrounding the promoter revealed possible binding sites for Mu proteins A and c, as well as for Escherichia coli integration host factor. Promoter fragments containing 5' and 3' deletions were fused to the lacZ reporter gene and assayed for activity after induction of a Mu prophage or a plasmid-borne mor gene. Sequences upstream of position -62 and downstream of +10 were dispensable for promoter activity. In DNase I footprinting with both crude extract and purified protein, Mor protected Pm sequences from position -56 to -33. Mutations disrupting the dyad symmetry of the terminator of early transcription overlapping the Mor binding site did not reduce promoter activity, suggesting that the symmetry per se is not required for Mor binding or Pm activation. Purified Mu lysogenic repressor (c) also bound to Pm, overlapping the Mor binding site. Production of large amounts of repressor in vivo reduced Mor-dependent promoter activity nearly 10-fold. Promoters with mutations in the repressor binding site showed a reduction in this repressor-mediated inhibition of Pm activity.
Analysis of CRP-CytR interactions at the Escherichia coli udp promoter
Authors: Brikun I , Suziedelis K , Stemmann O , Zhong R , Alikhanian L , Linkova E , Mironov A , Berg D E
Published: November 2016

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.
Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA
Authors: Costello G M , Vipond R , MacIntyre S
Published: November 2016

Two homologs of the outer membrane protein OmpA were identified in Aeromonas salmonicida by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and amino-terminal sequence analyses. An A. salmonicida genomic DNA library was constructed by using lambda GEM-11 and recombinant phage carrying both genes ompAI and ompAII) selected by immunoscreening. A 5.0-kb BamHI fragment containing the two genes in tandem was subcloned in pBluescript and used for further subcloning and sequencing of the genes. The encoded proteins (Mr = 33,564 and 32,536 for mature OmpAI and OmpAII, respectively) had only 64% identity with each other and otherwise had the highest level of homology to OmpA proteins from the members of the family Enterobacteriaceae. Based on the Escherichia coli OmpA model, an eight-stranded amphipathic beta-barrel model for the membrane assembly of the N-terminal half of OmpAI and OmpAII was predicted. Most variation between the two proteins was localized to the predicted surface loops and periplasmic turns, while the transmembrane strands and C-terminals domains were highly conserved. Expression of ompAI and ompAII separately in E. coli indicated that both genes could be independently transcribed from their own promoters and that both gene products were assembled into the E. coli outer membrane. A survey of different Aeromonas spp. by PCR revealed that possession of two tandem ompA genes was widespread among this genus. This is the first report of any bacterial species possessing two genes for homologs of this major outer membrane protein.
Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate
Authors: Cevallos M A , Encarnación S , Leija A , Mora Y , Mora J
Published: November 2016

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.
The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell cell envelope
Authors: Rodríguez-Herva J J , Ramos-Gonzalez M I , Ramos J L
Published: November 2016

Pseudomonas putida 14G-3, a derivative of the natural soil inhabitant P. putida KT2440, exhibited a chromosomal insertion of a mini-Tn5/'phoA transposon that resulted in reduced ability to colonize soil. In vitro characterization of P. putida 14G-3 revealed that it exhibited an altered cell morphology and envelope, as revealed by electron microscopy. The derived strain was sensitive to sodium dodecyl sulfate, deoxycholate, and EDTA, produced clumps when it reached high cell densities in the late logarithmic growth phase, and did not grow on low-osmolarity medium. The P. putida DNA surrounding the mini-Tn5/'phoA insertion was cloned and used as a probe to rescue the wild-type gene, which was sequenced. Comparison of the deduced peptide sequence with sequences in the Swiss-Prot database allowed the knocked-out gene to be identified as that encoding the peptidoglycan-associated lipoprotein (Pal or OprL) of P. putida. The protein was identified in coupled transcription and translation assays in vitro.
Transcriptional analysis of the 16s rRNA gene in Rickettsia prowazekii
Authors: Pang H , Winkler H H
Published: November 2016

The control of rRNA synthesis in the etiological agent of epidemic typhus, Rickettsia prowazekii, a slowly growing obligate intracytoplasmic bacterium, was investigated. Transcription of the rickettsial 16S rRNA gene (rrs), of which there is only a single copy, was controlled by a single promoter region, and the site for the initiation of transcription (base A) was found 117 bp upstream of the rrs coding region for the mature product. The promoter region contained an Escherichia coli promoter-like sequence, TTGACA-N17-TATAAC, centered 139 bp upstream of the coding region for the mature product. To investigate whether transcription of the rickettsial rrs responds to amino acid starvation conditions, total RNA was isolated from R. prowazekii-infected mouse L929 cells with or without methionine starvation. The level of newly synthesized 16S rRNA precursors in R. prowazekii, as analyzed by ribonuclease protection assays, decreased significantly after methionine starvation for 6 h and then recovered within 12 h after the addition of methionine. The chemical half-lives of the 16S rRNA precursors in the methionine-starved rickettsiae did not differ significantly from those in the normal rickettsiae. These results suggest that R. prowazekii regulates transcription of the rrs in response to amino acid starvation conditions.
Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation
Authors: Franklin M J , Ohman D E
Published: November 2016

Mucoid strains of Pseudomonas aeruginosa overproduce alginate, a linear exopolysaccharide Of D-mannuronate and variable amounts of L-guluronate. The mannuronate residues undergo modification by C-5 epimerization to form the L-guluronates and by the addition of acetyl groups at the 0-2 and 0-3 positions. Through genetic analysis, we previously identified algF, located upstream of algA in the 18-kb alginate biosynthetic operon, as a gene required for alginate acetylation. Here, we show the sequence of a 3.7-kb fragment containing the open reading frames termed algI, algJ, and algF. An algI::Tn5O1 mutant, which was defective in algIJFA because of the polar nature of the transposon insertion, produced alginate when algA was provided in trans. This indicated that the algIJF gene products were not required for polymer biosynthesis. To examine the potential role of these genes in alginate modification, mutants were constructed by gene replacement in which each gene (algI, algJ, or algF) was replaced by a polar gentamicin resistance cassette. Proton nuclear magnetic resonance spectroscopy showed that polymers produced by strains deficient in algIJF still contained a mixture of D-mannuronate and L-guluronate, indicating that C-5 epimerization was not affected. Alginate acetylation was evaluated by a colorimetric assay and Fourier transform-infrared spectroscopy, and this analysis showed that strains deficient in algIJF produced nonacetylated alginate. Plasmids that supplied the downstream gene products affected by the polar mutations were introduced into each mutant. The strain defective only in algF expression produced an alginate that was not acetylated, confirming previous results. Strains missing only algJ or algI also produced nonacetylated alginates. Providing the respective missing gene (algI, algJ, or algF) in trans restored alginate acetylation. Mutants defective in algI or algJ, obtained by chemical and transposon mutagenesis, were also defective in their ability to acetylate alginate. Therefore, algI and algJ represent newly identified genes that, in addition to algF, are required for alginate acetylation.
Analysis of the relationship between the decrease in pH and accumulation of 3-phosphoglyceric acid in developing forespores of Bacillus species
Authors: Magill N G , Cowan A E , Leyva-Vazquez M A , Brown M , Koppel D E , Setlow P
Published: November 2016

Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at approximately 4 h of sporulation preceded 3PGA accumulation, as observed previously in B. megaterium. These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B. subtilis, indicated that sigma G-dependent forespore transcription, but not sigma K-dependent mother cell transcription, is required for the forespore pH decrease. Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA. These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation. In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B. megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM approximately 10-fold in B. subtilis resulted in a large decrease in the spore's depot of 3PGA. The B. subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes.
Characterization of transmembrane domains 6, 7, and 8 of MalF by mutational analysis
Authors: Ehrle R , Pick C , Ulrich R , Hofmann E , Ehrmann M
Published: November 2016

Oligonucleotide mutagenesis was used to isolate mutations in membrane-spanning segments 6, 7, and 8 of MalF. MalF is a cytoplasmic membrane component of the binding protein-dependent maltose transport system in Escherichia coli. The current structural model predicts eight transmembrane domains for MalF. Membrane-spanning segments 6, 7, and 8 of MalF flank or are part of the EAA-X3-G-X9-I-X-LP consensus region present in the cytoplasmic membrane subunits of the bacterial ABC transporter superfamily members. Mutations with two novel phenotypes with respect to substrate specificity of the maltose transport system were isolated. One mutant grew on minimal maltose media but not on media containing either maltoheptaose or maltoheptaose plus maltose and was thus termed dextrin dominant negative. The other class of mutations led to a maltose minus but maltoheptaose plus phenotype. Nine of the isolated mutations leading to changes in substrate specificity were tightly clustered on one face of the postulated transmembrane helix 6. A similar clustering of mutations was detected in transmembrane domain 7. The majority of mutations in membrane-spanning segment 7 led to a protease-sensitive or a conditional phenotype with respect to MalF function or both. Mutations in transmembrane domain 8 appeared to be more randomly distributed. The majority of mutations in membrane-spanning segment 8 caused a Mal+ Dex- phenotype. Six Mal+ suppressor mutations isolated to two mutations in transmembrane domain 7 changed amino acid residues in membrane-spanning segment 6 or 8.
The proton/electron ration of the menaquinone-dependent electron transport from dihydrogen to tetrachloroethene in "Dehalobacter restrictus"
Authors: Schumacher W , Holliger C
Published: November 2016

In the anaerobic respiration chain of "Dehalobacter restrictus," dihydrogen functioned as the electron donor and tetrachloroethene (PCE) functioned as the electron acceptor. The hydrogenase faced the periplasm, and the PCE reductase faced the cytoplasmic side of the membrane. Both activities were associated with the cytoplasmic membrane. UV spectroscopy showed that membrane-bound menaquinone (MQ) was reduced by oxidation of H2 and reoxidized by reduction of PCE, indicating that MQ functions as an electron mediator. Fast proton liberation (t1/2 = 6 +/- 2 s) during electron transport from H2 to PCE and to trichloroethene (TCE) after addition of either PCE or TCE to H2-saturated cells resulted in an extrapolated H+/e- ratio of 1.25 +/- 0.2. This ratio indicated that besides the formation of protons upon oxidation of H2, vectorial translocation of protons from the inside to the outside could also occur. Proton liberation was inhibited by carbonylcyanide m-chlorophenylhydrazone (CCCP), 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO), and CuCl2. Fast proton liberation with an H+/e- ratio of 0.65 +/- 0.1 was obtained after addition of the MQ analog 2,3-dimethyl-1,4-naphthoquinone (DMN) as an oxidant pulse. This acidification was also inhibited by CCCP, HOQNO, and CuCl2. Oxidation of reduced DMN by PCE was not associated with fast acidification. The results with DMN indicate that the consumption and release of protons associated with redox reactions of MQ during electron transfer from H2 to PCE both occurred at the cytoplasmic side of the membrane. The PCE reductase was photoreversibly inactivated by 1-iodopropane, indicating that a corrinoid was involved in the PCE reduction.
Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth
Authors: Gallegos M T , Marqués S , Ramos J L
Published: November 2016

Growth of Pseudomonas putida (pWWO) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the XylS protein after binding of a benzoate effector. Alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xylS mediated by XylR activated by these compounds leads to overproduction of the XylS regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. We show here that in bacteria growing on glycerol or alkylbenzoates, the xylS gene is expressed at a low but constitutive level from a newly found sigma 70-dependent promoter called Ps2. The amount of XylS protein made from the transcript originated from Ps2 was sufficient to allow high levels of expression from the meta cleavage pathway operon promoter when the cells were grown in the presence of 3-methylbenzoate. The transcription initiation point of the transcript generated from Ps2 mapped 9 bp upstream from the proposed ATG of the xylS gene; this transcript contains the ribosome-binding site. The Ps2 promoter was located 110 bp downstream from a previously described sigma54-dependent promoter located upstream from the xylS open reading frame, now called Ps1. In cells growing on toluene or benzyl alcohols, the XylS regulator is overproduced as a consequence of increased expression of the gene through the effect of the two promoters working in tandem: the newly found sigma 70-dependent promoter, whose expression is XylR and toluene independent, and the sigma 54-dependent promoter, whose expression is dependent on XylR activated by its effectors. This expression pathway of the xylS gene explains why sigma 54-deficient P. putida bearing the wild-type TOL plasmid, or the wild-type P. putida strain bearing a TOL plasmid with a knocked-out xylR gene, can grow on alkylbenzoates. Until now this has been one of the unresolved paradoxes in the transcriptional control of the TOL meta cleavage pathway.
Cloning and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor in Vibrio alginolyticus
Authors: Okunishi I , Kawagishi I , Homma M
Published: November 2016

The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation for driving rotation of the helical flagellar filament. In this study, we cloned a gene (motY) encoding a component of the sodium-driven polar flagellar motor in Vibrio alginolyticus. Nucleotide sequence analysis revealed that the gene encodes a 293-amino-acid polypeptide with a single putative transmembrane segment that is very similar (94.5% identity) to the recently described MotY of V. parahaemolyticus. Their C-terminal domains were similar to the C-terminal domains of many peptidoglycan-interacting proteins, e.g., Escherichia coli MotB and OmpA, suggesting that MotY may interact with peptidoglycan for anchoring the motor. By using the lac promoter-repressor system, motY expression was controlled in V. alginolyticus cells. Swimming ability increased with increasing concentrations of the inducer isopropyl-beta-D-thiogalactopyranoside, and the swimming fraction increased after induction. These results are consistent with the notion that MotY is a component of the force-generating unit. V. alginolyticus motY complemented the motY mutation of V. parahaemolyticus. However, motY appeared to lack a region corresponding to the proposed motY promoter of V. parahaemolyticus. Instead, sequences similar to the sigma54 consensus were found in the upstream regions of both species. We propose that they are transcribed from the sigma54 -specific promoters.
Role of three chitin synthase genes in the growth of Candida albicans
Authors: Mio T , Yabe T , Sudoh M , Satoh Y , Nakajima T , Arisawa M , Yamada-Okabe H
Published: November 2016

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants.
A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles
Authors: Li Z , Clarke A J , Beveridge T J
Published: November 2016

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.
Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae
Authors: Bowen T L , Lin W C , Whitman W B
Published: November 2016

Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.