bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome
Authors: Rong L J , Karcher S J , Gelvin S B
Published: November 2016

picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A. tumefaciens. DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region. S1 nuclease mapping was used to identify the transcription initiation site of picA. Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A. tumefaciens, forming "ropes" in the presence of pea root cap cells. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts.
AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site
Authors: Mohr C D , Hibler N S , Deretic V
Published: November 2016

Strong transcriptional activation of algD, a key event in the overproduction of alginate and establishment of mucoidy in Pseudomonas aeruginosa, depends on the functional algR gene. The predicted gene product of algR shows homologies to response regulators from bacterial signal transduction systems. The algR gene was overexpressed in Escherichia coli, its product (AlgR) was purified by utilizing its apparent affinity for heparin, and its sequence was verified by partial amino acid sequence analysis. AlgR was found to interact directly with the algD promoter. Deletion mapping analysis, in conjunction with mobility shift DNA-binding assays, indicated the presence of three regions within the algD promoter capable of specifically binding AlgR. A relatively weak interaction was observed with the algD promoter fragment containing the region immediately upstream of the algD mRNA start site (-144 to +11). However, when fragments spanning regions located very far upstream from the algD mRNA initiation site (-533 and -332) were used, strong specific binding was observed. These regions were separated by a DNA segment not binding AlgR and spanning positions -332 to -144. DNase I footprinting analysis further established the presence of discrete AlgR binding sites overlapping with FUS, the far-upstream sites required for full induction of algD transcription and its environmental modulation. There were two distinct binding sites: RB1, spanning nucleotides -479 to -457, and RB2, spanning nucleotides -400 to -380. Both of these sequences shared a highly conserved core region, ACCGTTCGTC. These results established a direct interaction of AlgR with the algD promoter and revealed an arrangement of binding sites highly unusual for response regulators of the AlgR type.
Identification of the promoter region of the ribosome-releasing factor cistron (frr)
Authors: Shimizu I , Kaji A
Published: November 2016

Previous studies of the structure and expression of the ribosome-releasing factor (RRF) cistron (frr) have suggested that an efficient promoter region is located in the RRF cistron. We report here on the nucleotide sequence and in vivo function of the RRF promoter. The transcriptional start site was determined by primer extension to be 58 bp upstream of the translational initiation codon of frr. The location of the RRF promoter region was confirmed by means of (i) deletion analysis of the 5' proximal sequences of frr fused to the chloramphenicol acetyltransferase reporter gene, (ii) analysis of RRF produced in vivo from the deletion derivatives of frr cloned into pUC19, and (iii) gel retardation analysis with Escherichia coli RNA polymerase. The -35 and -10 regions were TTacCc and TATAcT, respectively. The strength of the RRF promoter was similar to that of the lac promoter, as determined by in vivo expression of chloramphenicol acetyltransferase activity. However, the RRF promoter was not affected by the intracellular cyclic AMP level despite the presence of a cyclic AMP receptor protein binding site downstream of the RRF promoter.
Transcriptional analysis of the Staphylococcus aureus plasmid pI258 mercury resistance determinant
Authors: Skinner J S , Ribot E , Laddaga R A
Published: November 2016

Northern blot DNA-RNA hybridization analysis of Staphylococcus aureus cells bearing pI258 showed that upon induction the amount of mer operon transcript present increased 49-fold over that observed in uninduced cells. Maximum induction occurred after 45 min in the presence of 5 microM HgCl2. Two transcripts, 5.0 and 5.8 kb long, were observed. Both transcripts encoded merR through merB (inclusive). Primer extension analysis determined that the 5' end of at least one transcript (and presumably of both) started at a T or G, 7 or 8 nucleotides downstream from the putative -10 site.
Cysteine, even in low concentrations, induces transient amino acid starvation in Escherichia coli
Authors: Sørensen M A , Pedersen S
Published: November 2016

Cysteine, in concentrations down to 0.04 micrograms/ml, induces transient amino acid starvation in Escherichia coli growing in minimal medium. The duration depends on the concentration and is 5 min at 2 micrograms of cysteine per ml. At low cysteine concentrations, threonine and isoleucine almost completely abolish the starvation.
Deletions or duplications in the BtuB protein affect its level in the outer membrane of Escherichia coli
Authors: Köster W , Gudmundsdottir A , Lundrigan M D , Seiffert A , Kadner R J
Published: November 2016

The Escherichia coli btuB product is an outer membrane protein that mediates the TonB-coupled active transport of cobalamins and the uptake of the E colicins and bacteriophage BF23. The roles of various segments of the BtuB protein in its function or cellular localization were investigated by analysis of several genetic constructs. Hybrid proteins in which various lengths from the amino terminus of BtuB were linked to alkaline phosphatase (btuB::phoA genes) were all secreted across the cytoplasmic membrane. The BtuB-PhoA proteins that carried up to 327 amino acids of BtuB appeared to reside in the periplasmic space, whereas hybrid proteins containing at least 399 amino acids of BtuB were associated with the outer membrane. Eleven in-frame internal deletion mutations that spanned more than half of the mature sequence were prepared by combining appropriate restriction fragments from btuB variants with 6-bp linker insertions. None of the deleted proteins was able to complement any BtuB functions, and only three of them were detectable in the outer membrane, suggesting that most of the deletions affected sequences needed for stable association with the outer membrane. Duplications covering the same portions of BtuB were prepared in the same manner. All of these partial duplication variants complemented all BtuB functions, although some gave substantially reduced levels of activity. These proteins were found in the outer membrane, although some were subject to proteolytic cleavage within or near the duplicated segment. These results indicate that the insertion of BtuB into the outer membrane requires the presence of several regions of teh BtuB protein and that the presence of extra or redundant segments of the protein can be tolerated during its insertion and function.
Arc and Sfr functions of the Escherichia coli K-12 arcA gene product are genetically and physiologically separable
Authors: Silverman P M , Rother S , Gaudin H
Published: November 2016

The Escherichia coli arcA gene product regulates chromosomal gene expression in response to deprivation of oxygen (Arc function; Arc stands for aerobic respiration control) and is required for expression of the F plasmid DNA transfer (tra) genes (Sfr function; Sfr stands for sex factor regulation). Using appropriate lacZ fusions, we have examined the relationship between these two genetic regulatory functions. Arc function in vivo was measured by anaerobic repression of a chromosomal sdh-lacZ operon fusion (sdh stands for succinate dehydrogenase). Sfr function was measured by activation of a plasmid traY-lacZ gene fusion. An eight-codon insertion near the 5' terminus of arcA, designated arcA1, abolished Arc function, as previously reported by S. Iuchi and E.C.C. Lin (Proc. Natl. Acad. Sci. USA 85:1888-1892, 1988), but left Sfr function largely (greater than or equal to 60%) intact. Similarly, the arcB1 mutation, which depressed sdh expression and is thought to act by abolishing the signal input that elicits ArcA function, had little effect (less than or equal to 20%) on the Sfr function of the arcA+ gene product. Conversely, a valine-to-methionine mutation at codon 203 (the sfrA5 allele) essentially abolished Sfr activity without detectably altering Arc activity. These data indicate that Sfr and Arc functions are separately expressed and regulated properties of the same protein.
Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli
Authors: Walderich B , Höltje J V
Published: November 2016

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.
An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production
Authors: Coucheron D H
Published: November 2016

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.
Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis
Authors: Black D S , Kelly A J , Mardis M J , Moyed H S
Published: November 2016

High-frequency persistence to the lethal effects of inhibition of either DNA or peptidoglycan synthesis, the Hip phenotype, results from mutations at the hip locus of Escherichia coli K-12. The nucleotide sequence of DNA fragments which complement these mutations revealed an operon consisting of a possible regulatory region, including sequences with modest homology to an E. coli promoter, and two open reading frames which are translated both in vitro and in vivo. The stop codon of a 264-bp open reading frame, hipB, and the start codon of a 1,320-bp open reading frame, hipA, share an adenine residue. Assays of promoter strength, the location of the probable promoter with respect to the start of transcription, and codon usage all indicate that hipB and hipA are weakly expressed genes. The activity of the promoter is impaired by an adjacent downstream sequence which includes the coding region of hipB. The impairment is partially relieved by insertion of a premature translation termination signal within the coding region of hipB, suggesting involvement of the HipB protein in the regulation of this promoter. The arrangement of hipB and hipA within the operon and the toxicity of hipA for strains defective in or lacking hipB suggest an important interaction between the products of these genes.
Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2
Authors: Krause M , Guiney D G
Published: November 2016

The Salmonella dublin virulence plasmid pSDL2 is a low-copy-number plasmid that is highly conserved in its host. Deletion of the 8-kb EcoRI C fragment downstream of the virulence region leads to plasmid instability and formation of multimers. We identified a multimer resolution system in the EcoRI C fragment composed of a trans-acting resolvase gene and a cis-acting resolution site. The resolvase gene, rsd, maps within a 2-kb EcoRV fragment and appears to be part of a multicistronic unit together with at least two other genes of unknown function. The derived protein, 28.7-kDa in size, is almost identical to the D protein of miniF. The C-terminal region was shown to have substantial similarity to the conserved C-terminal domains of the site-specific recombinases of the integrase family. The cis-acting resolution site, crs, is located upstream of rsd within a 628-bp SmaI-HpaI fragment. It contains eight direct incomplete 17-bp repeats followed by a segment rich in indirect repeats, the latter being homologous to the oriV1 sequence of miniF. crs contains the crossover site for specific recombination and mediates bidirectional promoter activity. A replicative function in analogy to that of oriV1 of F could not be demonstrated. The multimer resolution system was shown to stabilize pACYC184 and is dependent on the recA-mediated formation of multimeric plasmids. Screening different Salmonella serovars with a pSDL2-specific recombination assay revealed that only strains harboring a virulence plasmid encode for resolvase activity. Our results suggest that site-specific recombination contributes to the stable inheritance of pSDL2 and other Salmonella virulence plasmids.
Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype
Authors: Baird L , Lipinska B , Raina S , Georgopoulos C
Published: November 2016

We cloned and sequenced the sohB gene of Escherichia coli. The temperature-sensitive phenotype of bacteria that carry a Tn10 insertion in the htrA (degP) gene is relieved when the sohB gene is present in the cell on a multicopy plasmid (30 to 50 copies per cell). The htrA gene encodes a periplasmic protease required for bacterial viability only at high temperature, i.e., above 39 degrees C. The sohB gene maps to 28 min on the E. coli chromosome, precisely between the topA and btuR genes. The gene encodes a 39,000-Mr precursor protein which is processed to a 37,000-Mr mature form. Sequencing of a DNA fragment containing the gene revealed an open reading frame which could encode a protein of Mr 39,474 with a predicted signal sequence cleavage site between amino acids 22 and 23. Cleavage at this site would reduce the size of the processed protein to 37,474 Mr. The predicted protein encoded by the open reading frame has homology with the inner membrane enzyme protease IV of E. coli, which digests cleaved signal peptides. Therefore, it is possible that the sohB gene encodes a previously undiscovered periplasmic protease in E. coli that, when overexpressed, can partially compensate for the missing HtrA protein function.
Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction
Authors: Alam M , Hazelbauer G L
Published: November 2016

A number of eubacterial species contain methyl-accepting taxis proteins that are antigenically and thus structurally related to the well-characterized methyl-accepting chemotaxis proteins of Escherichia coli. Recent studies of the archaebacterium Halobacterium halobium have characterized methyl-accepting taxis proteins that in some ways resemble and in other ways differ from the analogous eubacterial proteins. We used immunoblotting with antisera raised to E. coli transducers to probe shared structural features of methyl-accepting proteins from archaebacteria and eubacteria and found substantial antigenic relationships. This implies that the genes for the contemporary methyl-accepting proteins are related through an ancestral gene that existed before the divergence of arachaebacteria and eubacteria. Analysis by immunoblot of mutants of H. halobium defective in taxis revealed that some strains were deficient in covalent modification of methyl-accepting proteins although the proteins themselves were present, while other strains appeared to be missing specific methyl-accepting proteins.
Constitutive and UV-mediated activation of RecA protein: combined effects of recA441 and recF143 mutations and of addition of nucleosides and adenine
Authors: Sassanfar M , Roberts J
Published: November 2016

The recF143 mutant of Escherichia coli is deficient in certain functions that also require the RecA protein: cell survival after DNA damage, some pathways of genetic recombination, and induction of SOS genes and temperate bacteriophage through cleavage of the LexA and phage repressors. To characterize the role of RecF in SOS induction and RecA activation, we determined the effects of the recF143 mutation on the rate of RecA-promoted cleavage of LexA, the repressor of the SOS genes. We show that RecA activation following UV irradiation is delayed by recF143 and that RecF is specifically involved in the SOS induction pathway that requires DNA replication. At 32 degrees C, the recA441 mutation partially suppresses the defect of recF mutants in inducing the SOS system in response to UV irradiation (A. Thomas and R. G. Lloyd, J. Gen. Microbiol. 129:681-686, 1983; M. R. Volkert, L. J. Margossian, and A. J. Clark, J. Bacteriol. 160:702-705, 1984); we find that this suppression occurs at the earliest detectable phase of LexA cleavage and does not require protein synthesis. Our results support the idea that following UV irradiation, RecF enhances the activation of RecA into a form that promotes LexA cleavage (A. Thomas and R. G. Lloyd, J. Gen. Microbiol. 129:681-686, 1983; M. V. V. S. Madiraju, A. Templin, and A. J. Clark, Proc. Natl. Acad. Sci. USA 85:6592-6596, 1988). In contrast to the constitutive activation phenotype of the recA441 mutant, the recA441-mediated suppression of recF is not affected by adenine and nucleosides. We also find that wild-type RecA protein is somewhat activated by adenine in the absence of DNA damage.
Nucleotide sequence of the gene (ard) encoding the antirestriction protein of plasmid colIb-P9
Authors: Delver E P , Kotova V U , Zavilgelsky G B , Belogurov A A
Published: November 2016

The IncI1 plasmid ColIb-P9 was found to encode an antirestriction function. The relevant gene, ard (alleviation of restriction of DNA), maps about 5 kb from the origin of transfer, in the region transferred early during bacterial conjugation. Ard inhibits both restriction and modification by each of the four type I systems of Escherichia coli tested, but it had no effect on restriction by either EcoRI, a type II system, or EcoP1, a type III system. The nucleotide sequence of the ColIb ard gene was determined; the predicted molecular weight of the Ard polypeptide is 19,193. The proposed polypeptide chain contains an excess of 25 negatively charged amino acids, suggesting that its overall character is very acidic. Deletion analysis of the gene revealed that the Ard protein contained a distinct functional domain located in the COOH-terminal half of the polypeptide. We suggest that the biological role of the ColIb Ard protein is associated with overcoming host-controlled restriction during bacterial conjugation.
Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1
Authors: Chistoserdov A Y , Tsygankov Y D , Lidstrom M E
Published: November 2016

An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization. Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present. Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known. The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene. The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.
In vitro activity of the nitrogen fixation regulatory protein FIXJ from Rhizobium meliloti
Authors: Batut J , Santero E , Kustu S
Published: November 2016

Cell extracts of an Escherichia coli strain that overproduces the regulatory protein FIXJ from Rhizobium meliloti promoted transcription of fixK, a known FIXJ-dependent gene, in a coupled transcription-translation assay. Activation by FIXJ was dependent on the sigma 70 holoenzyme form of RNA polymerase.
Dimethyl sulfoxide reductase of Escherichia coli: an investigation of function and assembly by use of in vivo complementation
Authors: Sambasivarao D , Weiner J H
Published: November 2016

Dimethyl sulfoxide (DMSO) reductase of Escherichia coli is a membrane-bound, terminal anaerobic electron transfer enzyme composed of three nonidentical subunits. The DmsAB subunits are hydrophilic and are localized on the cytoplasmic side of the plasma membrane. DmsC is the membrane-intrinsic polypeptide, proposed to anchor the extrinsic subunits. We have constructed a number of strains lacking portions of the chromosomal dmsABC operon. These mutant strains failed to grow anaerobically on glycerol minimal medium with DMSO as the sole terminal oxidant but exhibited normal growth with nitrate, fumarate, and trimethylamine N-oxide, indicating that DMSO reductase is solely responsible for growth on DMSO. In vivo complementation of the mutant with plasmids carrying various dms genes, singly or in combination, revealed that the expression of all three subunits is essential to restore anaerobic growth. Expression of the DmsAB subunits without DmsC results in accumulation of the catalytically active dimer in the cytoplasm. The dimer is thermolabile and catalyzes the reduction of various substrates in the presence of artificial electron donors. Dimethylnaphthoquinol (an analog of the physiological electron donor menaquinone) was oxidized only by the holoenzyme. These results suggest that the membrane-intrinsic subunit is necessary for anchoring, stability, and electron transport. The C-terminal region of DmsB appears to interact with the anchor peptide and facilitates the membrane assembly of the catalytic dimer.
A genetic analysis of Xis and FIS interactions with their binding sites in bacteriophage lambda
Authors: Numrych T E , Gumport R I , Gardner J F
Published: November 2016

The bacteriophage P22-based challenge-phage system was used to study the binding of Xis and FIS to their sites in attP of bacteriophage lambda. Challenge phages were constructed that contained the X1, X2, and F sites within the P22 Pant promoter, which is required for expression of antirepressor. If Xis and FIS bind to these sites in vivo, they repress transcription from Pant, allowing lysogenization to occur. Challenge phages carrying the XIX2F region in either orientation exhibited lysogenization dependent on both Xis and FIS. Neither Xis nor FIS was capable of functioning by itself as an efficient repressor in this system. This was the first time challenge phages have been constructed that require two different proteins bound simultaneously to act as a repressor. Mutations in the X1, X2, and F sites that inhibit Xis and FIS from binding were isolated by selecting mutant phages that still expressed antirepressor synthesis in the presence of Xis and FIS. DNA sequence analysis of the mutants revealed 38 unique mutations, including single-base-pair substitutions, multiple-base-pair changes, deletions, and insertions throughout the entire X1, X2, and F regions. Some of the mutations verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, while others provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them.
Phylogenetic analysis of the spirochetes
Authors: Paster B J , Dewhirst F E , Weisburg W G , Tordoff L A , Fraser G J , Hespell R B , Stanton T B , Zablen L , Mandelco L , Woese C R
Published: November 2016

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. THe borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated fromt the shew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema illini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.
Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans
Authors: Masters C I , Smith M D , Gutman P D , Minton K W
Published: November 2016

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.
Phosphatidylglycerolphosphate synthase expression in Schizosaccharomyces pombe is regulated by the phospholipid precursors inositol and choline
Authors: Karkhoff-Schweizer R R , Kelly B L , Greenberg M L
Published: November 2016

The enzyme phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the committed step in the cardiolipin biosynthetic pathway. To study the regulation of PGPS in Schizosaccharomyces pombe, we characterized the enzyme biochemically. Maximum activity occurred in the presence of 6 mM Triton X-100 at pH 7.5. The apparent Km values for CDP-diacylglycerol and glycerol 3-phosphate were 130 and 26 microM, respectively. Optimal activity was at 35 degrees C, and enzyme activity was labile above 40 degrees C. Thioreactive agents were inhibitory to PGPS activity. To determine whether S. pombe PGPS is regulated by phospholipid precursors, we examined the time-dependent expression of PGPS upon inositol and choline starvation. Starvation for inositol resulted in a threefold increase in PGPS expression in wild-type cells. In cho1 and cho2 mutants, which are blocked in phosphatidylcholine synthesis, starvation for choline resulted in transient derepression of PGPS expression. In choline auxotrophs starved for inositol, PGPS was derepressed 2.5- to 3-fold in the presence of choline and less or not at all in the absence of choline. This is the first description of PGPS regulation in S. pombe and the first demonstration of inositol-mediated regulation in the inositol-requiring yeast species.
Isolation and characterization of a new class of cytochrome d terminal oxidase mutants of Escherichia coli
Authors: Oden K L , Gennis R B
Published: November 2016

Cytochrome d terminal oxidase mutants were isolated by using hydroxylamine mutagenesis of pNG2, a pBR322-derived plasmid containing the wild-type cyd operon. The mutagenized plasmid was transformed into a cyo cyd recA strain, and the transformants were screened for the inability to confer aerobic growth on nonfermentable carbon sources. Western blot analysis and visible-light spectroscopy were performed to characterize three independent mutants grown both aerobically and anaerobically. The mutational variants of the cytochrome d complex were stabilized under anaerobic growth conditions. All three mutations perturb the b595 and d heme components of the complex. These mutations were mapped and sequenced and are shown to be located in the N-terminal third of subunit II of the cytochrome d complex. It is proposed that the N terminus of subunit II may interact with subunit I to form an interface that binds the b595 and d heme centers.
Electrogenic L-malate transport by Lactobacillus plantarum: a basis for energy derivation from malolactic fermentation
Authors: Olsen E B , Russell J B , Henick-Kling T
Published: November 2016

L-Malate transport in Lactobacillus plantarum was inducible, and the pH optimum was 4.5. Malate uptake could be driven by an artificial proton gradient (delta pH) or an electroneutral lactate efflux. Because L-lactate efflux was unable to drive L-malate transport in the absence of a delta pH, it did not appear that the carrier was a malate-lactate exchanger. The kinetics of malate transport were, however, biphasic, suggesting that the external malate concentration was also serving as a driving force for low-affinity malate uptake. Because the electrical potential (delta psi, inside negative) inhibited malate transport, it appeared that the malate transport-lactate efflux couple was electrogenic (net negative) at high concentrations of malate. De-energized cells that were provided with malate only generated a large proton motive force (greater than 100 mV) when the malate concentration was greater than 5 mM, and malate only caused an increase in cell yield (glucose-limited chemostats) when malate accumulated in the culture vessel. The use of the malate gradient to drive malate transport (facilitated diffusion) explains how L. plantarum derives energy from malolactic fermentation, a process which does not involve substrate-level phosphorylation.
Dissolution and immunochemical analysis of the sheath of the archaeobacterium Methanospirillum hungatei GP1
Authors: Southam G , Beveridge T J
Published: November 2016

The sheath of Methanospirillum hungatei GP1 was degraded by three dissolution techniques, which produced a range of soluble products. By using 0.05 M L-arginine buffer (pH 12.6) at 90 degrees C for 10 min, 74% (dry weight) of the sheath was dissolved; however, the solubilized polypeptides were extensively degraded. Treatment with 2% beta-mercaptoethanol and 2% sodium dodecyl sulfate at 90 degrees C in 0.05 M 2(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 9.0) solubilized 42% (dry weight) of the sheath as a group of polypeptides of 30 to 40 kDa. At 100 degrees C for 2 h, 5% beta-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and 20 mM EDTA released 74% of the sheath's mass as a group of polypeptides of 10 to 40 kDa. All solubilized products were examined by SDS-polyacrylamide gel electrophoresis, and a range of high- and low-molecular-weight polypeptides was identified. None were glycoproteins. Hoops, which comprise the sheath's structure, were seen by electron microscopy after all of the attempted dissolutions. Monoclonal antibodies were produced against the 10- to 40-kDa range of solubilized products and against the approximately 40-kDa polypeptides, and polyclonal antiserum was produced against an 18-kDa polypeptide. These immunological markers were used in Western immunoblotting and protein A-colloidal gold-antibody probing by electron microscopy to identify the structural location of the various polypeptides. Native sheath, which possesses 2.8-nm particles on its outer surface (M. Stewart, T.J. Beveridge, and G.D. Sprott, J. Mol. Biol. 183:509-515, 1985; P.J. Shaw, G.J. Hills, J.A. Henwood, J.E. Harris, and D.B. Archer, J. Bacteriol. 161:750-757, 1985), presented a gentle wave-form surface in platinum-shadowed specimens. In contrast, the inner face of the sheath was highlighted by ridges lying perpendicular to the longitudinal axis of the sheath and likely corresponded to hoop boundaries. Both the polyclonal and monoclonal antibodies were specific for different faces; polyclonal antibodies labeled the inner face, whereas monoclonal antibodies labeled the outer face. Accordingly, the apparent asymmetry of structure between the two faces of the sheath can be correlated by our immunochemical probing with a distinct asymmetry in the distribution of exposed polypeptides between the faces. The possible implications of this asymmetry for growth and maturation of the sheath are explained.
Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli
Authors: Tobin M B , Kovacevic S , Madduri K , Hoskins J A , Skatrud P L , Vining L C , Stuttard C , Miller J R
Published: November 2016

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete.
The Escherichia coli ts8 mutation is an allele of fda, the gene encoding fructose-1,6-diphosphate aldolase
Authors: Singer M , Rossmiessl P , Cali B M , Liebke H , Gross C A
Published: November 2016

The ts8 mutant of Escherichia coli has previously been shown to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive temperature. We demonstrate in this report that the ts8 mutation is an allele of fda, the gene that encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase. We show that ts8 and a second fda mutation, h8, isolated and characterized by A. Böck and F. C. Neidhardt, are dominant mutations and that they encode a thermolabile aldolase activity.
Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior
Authors: Belas R , Erskine D , Flaherty D
Published: November 2016

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed.
Genes coding for integration host factor are conserved in gram-negative bacteria
Authors: Haluzi H , Goitein D , Koby S , Mendelson I , Teff D , Mengeritsky G , Giladi H , Oppenheim A B
Published: November 2016

A genetic system for the selection of clones coding for integration host factor and HU homologs is described. We demonstrate that the himA and hip genes of Serratia marcescens and Aeromonas proteolytica can substitute for the Escherichia coli genes in a variety of biological assays. We find that the sequence and genetic organization of the himA and hip genes of S. marcescens are highly conserved.
Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans
Authors: Debussche L , Couder M , Thibaut D , Cameron B , Crouzet J , Blanche F
Published: November 2016

Cob(I)alamin adenosyltransferase (EC 2.5.1.17) was purified to homogeneity from extracts of a Pseudomonas denitrificans recombinant strain and sequenced at its N terminus. It is a homodimer (each unit with an Mr of 28,000) encoded by cobO. The enzyme adenosylated all of the corrinoids isolated from this microorganism but did not adenosylate cobyrinic acid.
Membrane localization of the HflA regulatory protease of Escherichia coli by immunoelectron microscopy
Authors: Zorick T S , Echols H
Published: November 2016

The hflA locus of Escherichia coli specifies a multisubunit protease that selectively degrades the cII transcriptional activator of phage lambda. The regulated turnover of cII is critical for the choice between the lytic and lysogenic pathways of viral development. Previous cell fractionation work has indicated that HflA is associated with the inner membrane fraction. We have sought to demonstrate that the HflA protease is localized in the cell membrane of intact cells. To achieve this goal, we have combined electron microscopy of thin-sectioned E. coli cells with antibody tagging by a colloidal gold label. Using antibody to purified HflA protein, we have found preferential membrane labeling for hflA+ cells but not for hflA mutant cells. We conclude that HflA protease is localized in the cell membrane. The membrane location for HflA protein may serve as a component of a targeting mechanism to limit the action of the regulatory protease to selected cytoplasmic proteins.
Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions
Authors: Reuber T L , Long S , Walker G C
Published: November 2016

The exo loci of Rhizobium meliloti are necessary for the production of an acidic exopolysaccharide, EPS I, that is needed for alfalfa nodule invasion by strain Rm1021. We have isolated and characterized alkaline phosphatase fusions made with TnphoA in several exo loci of R. meliloti and used these fusions to examine the subcellular localization of exo gene products and the regulation of exo genes in free-living cells and in planta. In the course of this work, we isolated a new exo locus, exoT. We have obtained evidence that several of the exo loci may encode membrane proteins. The activity of TnphoA fusions in several exo loci is increased two- to fivefold in the presence of the regulatory mutations exoR95 and exoS96. While examining the regulation of the exo gens by exoR95 and exoS96, we found that certain classes of exo mutations are lethal in an exoR95 or exoS96 background unless a plasmid complementing the exo mutation is present. This result has possible implications for the role of these exo loci in EPS I biosynthesis. We have developed a method for staining nodules specifically for the alkaline phosphatase activity present in the inducing bacteria and used this method to show that an exoF::TnphoA fusion is expressed mainly in the invasion zone of the nodule.
Plasmid-mediated sucrose metabolism in Escherichia coli: characterization of scrY, the structural gene for a phosphoenolpyruvate-dependent sucrose phosphotransferase system outer membrane porin
Authors: Hardesty C , Ferran C , DiRienzo J M
Published: November 2016

The scrY gene, part of the pUR400-borne sucrose regulon, appeared to be transcribed from its own promoter, with the transcriptional start site located 58 bp upstream from the initiation codon. An open reading frame encoding a polypeptide of 505 amino acid residues (Mr 55,408) was identified. The first 22 amino acid residues formed a leader sequence typical of those found in other procaryotic outer membrane and periplasmic proteins. A frameshift mutation in the scrY gene resulted in a dramatic decrease in sucrose transport with no effect on in vitro phosphorylation activity associated with enzyme IISer. The rate of diffusion of sucrose was 96 times greater than the rate of diffusion of lactose or maltose in liposomes containing the ScrY protein. This increase in sucrose permeability provided strong evidence that the ScrY protein functions as a sucrose porin. There was 23% amino acid sequence identity between the ScrY protein and LamB, a maltose porin from Escherichia coli.