bluuebunny/crossref_metadata_2025 huggingface dataset:
Phylogeny of Sym plasmids of rhizobia by PCR-based sequencing of a nodC segment
Authors: Ueda T , Suga Y , Yahiro N , Matsuguchi T
Published: November 2016

To understand the host specificity of rhizobia and the relationship between the evolution of Sym plasmids and that of host plants, we determined partial nodC sequences of 10 representative rhizobium strains and then constructed an evolutionary tree for the deduced amino acid sequences with four published sequences. These coding sequences yield a phylogenetic tree similar to that for leghemoglobin of host plants, suggesting that the evolution of common nodulation genes may be linked to host legume evolution and speciation.
Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens
Authors: Meurer G , Hutchinson C R
Published: November 2016

The significance of potential active site motifs for acyltransferase and beta-ketoacyl:acyl carrier protein synthase regions within the TcmK protein was investigated by determining the effects of mutations in the proposed active sites on the production of tetracenomycins F2 and C. In a Streptomyces glaucescens tcmGHI JKLMNO null mutant, plasmids carrying the S351A mutation produced high amounts of tetracenomycin F2 but plasmids carrying the C173A or C173S mutation or the H350L-S351A double mutation produced no detectable amount of any known intermediate. In a tcmK mutant, plasmids with the S351A mutation restored high production of tetracenomycin C and plasmids carrying the other mutations were able to complement the chromosomal defect to some extent. None of the mutations affected the amount of TcmK produced.
Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae
Authors: Castro C , Ribas J C , Valdivieso M H , Varona R , del Rey F , Duran A
Published: November 2016

Papulacandin B, an antifungal agent that interferes with the synthesis of yeast cell wall (1,3)beta-D-glucan, was used to isolate resistant mutants in Schizosaccharomyces pombe and Saccharomyces cerevisiae. The resistance to papulacandin B always segregated as a recessive character that defines a single complementation group in both yeasts (pbr1+ and PBR1, respectively). Determination of several kinetic parameters of (1,3)beta-D-glucan synthase activity revealed no differences between S. pombe wild-type and pbr1 mutant strains except in the 50% inhibitory concentration for papulacandin B of the synthases (about a 50-fold increase in mutant activity). Inactivation of the synthase activity of both yeasts after in vivo treatment with the antifungal agent showed that mutant synthases were more resistant than the corresponding wild-type ones. Detergent dissociation of the S. pombe synthase into soluble and particulate fractions and subsequent reconstitution indicated that the resistance character of pbr1 mutants resides in the particulate fraction of the enzyme. Cloning and sequencing of PBR1 from S. cerevisiae revealed a gene identical to others recently reported (FKS1, ETG1, CWH53, and CND1). Its disruption leads to reduced levels of both (1,3)beta-D-glucan synthase activity and the alkali-insoluble cell wall fraction. Transformants containing the PBR1 gene reverse the defect in (1,3)beta-D-glucan synthase. It is concluded that Pbr1p is probably part of the (1,3)beta-D-glucan synthase complex.
Nucleotide sequence, transcriptional analysis, and glucose regulation of the phenoxazinone synthase gene (phsA) from Streptomyces antibioticus
Authors: Hsieh C J , Jones G H
Published: November 2016

The nucleotide sequence of a 2.3-kb SphI fragment containing the structural gene (phsA) for phenoxazinone synthase (PHS) of Streptomyces antibioticus was determined. The sequence was found to contain an open reading frame (ORF) with a G+C content of 71.5% oriented in the direction of transcription that was confirmed by primer extension. The ORF encodes a protein with an M(r) of 70,223 consisting of 642 amino acids and is preceded by a potential ribosome-binding site. The codon usage pattern is in agreement with the general pattern for streptomycete genes, with a 92.5 mol% G+C content in the third position. The N-terminal sequence of the mature PHS subunit corresponds exactly to that predicted from the nucleotide sequence. Neither ATG nor GTG initiator codons were identified for the protein. However, a TTG codon was located near the amino terminus of the mature protein and is a good candidate for the initiator codon. The transcriptional start point of phsA was located 36 bp upstream of the start codon by primer extension. The -10 region of the putative promoter showed some similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 region was less similar. Comparison of the primary amino acid sequence of PHS of S. antibioticus with other amino acid sequences indicated that PHS is a blue copper protein with copper binding domains in the N-terminal and C-terminal regions of the polypeptide chain. A BsrBI fragment containing the promoter region of phsA and a portion of the ORF was shown to promote xylE expression when cloned in the streptomycete promoter probe vector pIJ2843. This phsA promoter-dependent xylE expression could be repressed by glucose in S. antibioticus when the organism was grown on glucose or galactose plus glucose. Thus, the cloned promoter region appears to contain the sequences responsible for catabolite repression of PHS production.
Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad
Authors: Massad G , Zhao H , Mobley H L
Published: November 2016

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.
catM encodes a LysR-type transcriptional activator regulating catechol degradation in Acinetobacter calcoaceticus
Authors: Romero-Arroyo C E , Schell M A , Gaines G L , Neidle E L
Published: November 2016

On the basis of the constitutive phenotypes of two catM mutants of Acinetobacter calcoaceticus, the CatM protein was proposed to repress expression of two different loci involved in catechol degradation, catA and catBCIJFD (E. Neidle, C. Hartnett, and L. N. Ornston, J. Bacteriol. 171:5410-5421, 1989). In spite of its proposed negative role as a repressor, CatM is similar in amino acid sequence to positive transcriptional activators of the LysR family. Investigating this anomaly, we found that insertional inactivation of catM did not cause the phenotype expected for the disruption of a repressor-encoding gene: in an interposon-generated catM mutant, no cat genes were expressed constitutively, but rather catA and catB were still inducible by muconate. Moreover, this catM mutant grew poorly on benzoate, a process requiring the expression of all cat genes. The inducibility of the cat genes in this catM mutant was completely eliminated by a 3.5-kbp deletion 10 kbp upstream of catM. In this double mutant, catM in trans restored muconate inducibility to both catA and catB. These results suggested the presence of an additional regulatory locus controlling cat gene expression. The ability of CatM to function as an activator was also suggested by these results. In support of this hypothesis, in vivo methylation protection assays showed that CatM protects two guanines in a dyad 65 nucleotides upstream of the catB transcriptional start site, in a location and pattern typical of LysR-type transcriptional activators. Gel mobility shift assays indicated that CatM also binds to a region upstream of catA. DNA sequence analysis revealed a nucleotide near the 3' end of catM not present in the published sequence. Translation of the corrected sequence resulted in the deduced CatM protein being 52 residues longer than previously reported. The size, amino acid sequence, and mode of action of CatM now appear similar to, and typical of, what has been found for transcriptional activators in the LysR family. Analysis of one of the constitutive alleles of catM previously thought to encode a dysfunctional repressor indicated instead that it encodes an inducer-independent transcriptional activator.
Synthesis of sn-glycerol 3-phosphate, a key precursor of membrane lipids, in Bacillus subtilis
Authors: Morbidoni H R , de Mendoza D , Cronan J E
Published: November 2016

The Bacillus subtilis gpsA gene was cloned by complementation of an Escherichia coli gpsA strain auxotrophic for sn-glycerol 3-phosphate. The gene was sequenced and found to encode an NAD(P)H-dependent dihydroxyacetone phosphate reductase with a deduced molecular mass of 39.5 kDa. The deduced amino acid sequence showed strong conservation with that of the E. coli homolog and to other procaryotic and eucaryotic dihydroxyacetone phosphate reductases. The physical location of gpsA on the B. subtilis chromosome was at about 200 degrees. Disruption of the chromosomal gpsA gene yielded B. subtilis strains auxotrophic for glycerol, indicating that the gpsA gene product is responsible for synthesis of the sn-glycerol 3-phosphate required for phospholipid synthesis. We also found that transformation of the classical B. subtilis glycerol auxotrophs with a gpsA-containing genomic fragment yielded transformants that grew in the absence of glycerol. In agreement with prior work, our attempts to determine the reductase activity in B. subtilis extracts were unsuccessful. However, expression of the B. subtilis gpsA gene in E. coli gave reductase activity that was only slightly inhibited by sn-glycerol 3-phosphate. Since the E. coli GpsA dihydroxyacetone phosphate reductase is very sensitive to allosteric inhibition by sn-glycerol 3-phosphate, these results indicate that the B. subtilis gpsA-encoded reductase differs from that of E. coli. It seems that B. subtilis regulates sn-glycerol 3-phosphate synthesis at the level of gene expression rather than through the E. coli mechanism of strong allosteric inhibition of an enzyme produced in excess.
Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis
Authors: Webb C D , Decatur A , Teleman A , Losick R
Published: November 2016

We report the use of the green fluorescent protein (GFP) of Aequorea victoria to visualize cell-specific gene expression and protein subcellular localization during sporulation in Bacillus subtilis. Sporangia bearing the gene (gfp) for the green fluorescent protein fused to genes under the control of the sporulation transcription factor sigma F exhibited a forespore-specific pattern of fluorescence. Forespore-specific fluorescence could be detected with fusions to promoters that are utilized with low (csfB) and high (sspE-2G) efficiency by sigma F-containing RNA polymerase. Conversely, a mother cell-specific pattern of fluorescence was observed in sporangia bearing a transcriptional fusion of gfp to a spore coat protein gene (cotE) under the control of sigma E and an in-frame fusion to a regulatory gene (gerE) under the control of sigma K. An in-frame fusion of gfp to cotE demonstrated that GFP can also be used to visualize protein subcellular localization. In sporangia producing the CotE-GFP fusion protein, fluorescence was found to localize around the developing spore, and this localization was dependent upon SpoIVA, a morphogenetic protein known to determine proper localization of CotE.
Molecular cloning and characterization of an adherence-related operon of Mycoplasma genitalium
Authors: Reddy S P , Rasmussen W G , Baseman J B
Published: November 2016

Adhesins and adhesin-related accessory proteins of pathogenic mycoplasmas are required for cytadherence and the subsequent development of disease pathology. The classic example has been Mycoplasma pneumoniae, which causes primary atypical pneumonia in humans. Mutants of M. pneumoniae defective in adhesins (P1 and P30) or in adherence-accessory proteins (HMW1 through HMW4) are unable to colonize host tissues and are avirulent. Mycoplasma genitalium, implicated in nongonococcal, nonchlamydial urethritis, pneumonia, arthritis, and AIDS progression, was found to encode a 140-kDa adhesin that shared both DNA and protein sequence similarities with P1, a major adhesin of M. pneumoniae. In this report, we show that M. genitalium possesses additional homolog sequences to well-characterized adherence-related genes and proteins of M. pneumoniae. The M. genitalium homologs are designated P32 and P69 and correspond to P30 and HMW3 of M. pneumoniae, respectively (J. B. Baseman, p. 243-259, in S. Rottem and I. Kahane, ed., Subcellular biochemistry, vol. 20. Mycoplasma cell membranes, 1993, and D. C. Krause, D. K. Leith, R. M. Wilson, and J. B. Baseman, Infect. Immun. 35:809-817, 1982). Interestingly, the operon-like organizations of P32 and P69 in the M. genitalium genome are similar to the organizations of P30 and HMW3 genes of M. pneumoniae, suggesting that the conservation of these adherence-related genes and proteins might have occurred through horizontal gene transfer events originating from an ancestral gene family.
CheA, CheW, and CheY are required for chemotaxis to oxygen and sugars of the phosphotransferase system in Escherichia coli
Authors: Rowsell E H , Smith J M , Wolfe A , Taylor B L
Published: November 2016

We carried out studies with Escherichia coli to determine the site at which the methylation-independent pathways for taxis to oxygen and to sugars of the phosphoenolpyruvate:sugar phosphotransferase transport system converge with the methylation-dependent chemotaxis pathways. Using genetic reconstitution of the pathways in a null strain, we determined that all pathways examined required the products of the genes cheA, cheW, and cheY. Thus, we conclude that both the methylation-independent and methylation-dependent pathways converge at CheA, the histidine kinase product of cheA.
Distance between alleles as a determinant of linkage in natural transformation of Acinetobacter calcoaceticus
Authors: Kloos D U , DiMarco A A , Elsemore D A , Timmis K N , Ornston L N
Published: November 2016

Cotransformation frequencies of 16, 39, 51, and 60% were observed when donor alleles were separated by distances of 9.2, 7.4, 6.3, and 5.1 kb, respectively, in donor Acinetobacter calcoaceticus DNA. A different and unexpected pattern was observed when the distance between recipient alleles was reduced from 9.2 to 5.1 kb. Ligation of unlinked chromosomal DNA fragments allowed them to be linked genetically through natural transformation.
A DNA segment conferring stable maintenance on R6K gamma-origin core replicons
Authors: Wu F , Levchenko I , Filutowicz M
Published: November 2016

The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.
Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein
Authors: Keuntje B , Masepohl B , Klipp W
Published: November 2016

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.
Specific binding of PapI to Lrp-pap DNA complexes
Authors: Kaltenbach L S , Braaten B A , Low D A
Published: November 2016

Expression of pyelonephritis-associated pili (Pap) varies between transcriptionally active (ON) and inactive (OFF) phase states. Pap phase variation is controlled by the binding of leucine-responsive regulatory protein (Lrp) to two pap regulatory DNA regions, each containing a deoxyadenosine methylase site and designated GATC-I and GATC-II. Methylation of these GATC sites modulates binding of Lrp and plays an essential role in phase variation. PapI, an 8.8-kDa pap-encoded regulatory protein, plays a key role in the switch between OFF and ON transcription states. In the absence of PapI, Lrp binds to sites overlapping the papBA promoter and inhibits transcription. Addition of PapI results in a translocation of Lrp binding to sites over 100 bp upstream, resulting in the ON transcription state. Gel shift analysis using radiolabeled PapI shows that PapI binds with high specificity to Lrp-pap DNA complexes but binds only weakly to free Lrp. Protein cross-linking studies indicate that Lrp and PapI directly interact with each other. On the basis of these data, we present a hypothesis in which PapI facilitates the transition between OFF and ON transcription states by binding to Lrp and altering Lrp's affinity for the pap GATC-I and GATC-II regions.
Organization and transcriptional regulation of the Escherichia coli K-12 D-serine tolerance locus
Authors: Nørregaard-Madsen M , McFall E , Valentin-Hansen P
Published: November 2016

We have reinvestigated the genetic organization and the transcription regulation of the dsd operon of Escherichia coli. By combining genetic and biochemical studies, it is demonstrated that the regulatory region of the operon and the gene encoding the specific regulator of D-serine tolerance (dsdC) had been misplaced in previous work on the dsd system. Also, the previous erroneous DNA sequence of the dsdC gene has been corrected. It turned out that an additional gene (dsdX) is present immediately upstream of dsdA (encoding D-serine deaminase) and that dsdC is located adjacent to dsdX. The dsdXA genes are cotranscribed from a common promoter region present in the dsdX-dsdC intercistronic region. The DsdC activator belongs to the LysR-type of transcriptional regulators and is absolutely required for dsdA expression. Additionally, the activity of the dsdXA promoter depends on the cyclic AMP receptor protein, and the two activators act in concert to synergistically activate transcription.
Physical map of the linear chromosome of Streptomyces griseus
Authors: Lezhava A , Mizukami T , Kajitani T , Kameoka D , Redenbach M , Shinkawa H , Nimi O , Kinashi H
Published: November 2016

The chromosomal DNA of Streptomyces griseus 2247 (a derivative of strain IFO3237) was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestion with AseI and DraI gave 15 and 9 fragments, respectively, the total sizes of which were 7.8 Mb. All the AseI and DraI fragments were aligned on a linear chromosome map by using linking plasmids and cosmids. PFGE analysis of the intact chromosome also showed a linear DNA band of about 8 Mb. Detailed physical maps of both terminal regions were constructed; they revealed the presence of a 24-kb terminal inverted repeat on each end. PFGE analysis with and without proteinase K treatment suggested that each end of the chromosome carries a protein molecule.
Identification and functional analysis of the transfer region of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica
Authors: Vrijbloed J W , van der Put N M , Dijkhuizen L
Published: November 2016

Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) that is present either as an integrated element or as an autonomously replicating plasmid. Conjugational transfer of pMEA300 results in pock formation, zones of growth inhibition that become apparent when plasmid-carrying donor cells develop in a confluent lawn of plasmid-lacking recipient cells. A 6.2-kb pMEA300 DNA region specifying the functions of conjugation and pock formation was sequenced, revealing 10 open reading frames. This is the first sequence of the transfer region of a plasmid from a nonstreptomycete actinomycete. No clear similarities were found between the deduced sequences of the 10 putative Tra proteins of pMEA300 and those of Streptomyces plasmids. All Tra proteins of pMEA300 thus may represent unfamiliar types. A detailed mutational analysis showed that at least four individual proteins, TraG (9,488 Da), TraH (12,586 Da), TraI (40,468 Da), and TraJ (81,109 Da), are required for efficient transfer of pMEA300. Their disruption resulted in a clear reduction in the conjugational transfer frequencies, ranging from (5.2 x 10(1))-fold (TraG) to (2.3 x 10(6))-fold (TraJ), and in reduced pock sizes. At least two putative proteins, TraA (10,698 Da) and TraB (31,442 Da), were shown to be responsible for pock formation specifically. Specific binding of the pMEA300-encoded KorA protein to the traA-korA intragenic region was observed.
Benzoate-coenzyme A ligase, encoded by badA, is one of three ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth of Rhodopseudomonas palustris on benzoate
Authors: Egland P G , Gibson J , Harwood C S
Published: November 2016

The first step of anaerobic benzoate degradation is the formation of benzoyl-coenzyme A by benzoate-coenzyme A ligase. This enzyme, purified from Rhodopseudomonas palustris, is maximally active with 5 microM benzoate. To study the molecular basis for this reaction, the benzoate-coenzyme A ligase gene (badA) was cloned and sequenced. The deduced amino acid sequence of badA showed substantial similarity to other coenzyme A ligases, with the highest degree of similarity being that to 4-hydroxybenzoate-coenzyme A ligase (50% amino acid identity) from R. palustris. A badA mutant that was constructed had barely detectable levels of ligase activity when cell extracts were assayed at 10 microM benzoate. Despite this, the mutant grew at wild-type rates on benzoate under laboratory culture conditions (3 mM benzoate), and mutant cell extracts had high levels of ligase activity when assayed at a high concentration of benzoate (1 mM). This suggested that R. palustris expresses, in addition to BadA, a benzoate-activating enzyme(s) with a relatively low affinity for benzoate. A possible role of 4-hydroxybenzoate-coenzyme A ligase (encoded by hbaA) in this capacity was investigated by constructing a badA hbaA double mutant. Although the double mutant grew more slowly on benzoate than badA cells, growth rates were still significant, suggesting the involvement of a third enzyme in benzoate activation. Competition experiments involving the addition of a small amount of cyclohexanecarboxylate to ligase assay mixtures implicated cyclohexanecarboxylate-coenzyme A ligase as being this third enzyme. These results show that wild-type R. palustris cells synthesize at least three enzymes that can catalyze the initial step in anaerobic benzoate degradation during growth on benzoate. This observation supports previous suggestions that benzoyl-coenzyme A formation plays a central role in anaerobic aromatic compound biodegradation.
The chromosome of Salmonella paratyphi A is inverted by recombination between rrnH and rrnG
Authors: Liu S L , Sanderson K E
Published: November 2016

Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever. We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca. 4,600 kb. With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S. paratyphi A ATCC 9150 and located these insertions on the S. paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10. Compared with the maps of other Salmonella species, the S. paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes. We propose that during the evolution of S. paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance. The insertion and the inversion are both present in all 10 independent wild-type S. paratyphi A strains tested.
1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium
Authors: Li J N , Björk G R
Published: November 2016

1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.
Ultrastructural diversity of the cellulase complexes of Clostridium papyrosolvens C7
Authors: Pohlschröder M , Canale-Parola E , Leschine S B
Published: November 2016

Transmission electron microscopy was used to investigate the ultrastructural features of diverse cellulase and cellulase-xylanase multiprotein complexes that are components of the cellulase-xylanase system of Clostridium papyrosolvens C7. The multiprotein complexes were separated by anion-exchange chromatography into seven biochemically distinguishable fractions (F1 to F7). Most individual F fractions contained, in relatively large numbers, an ultrastructurally recognizable type of particle that occurred only in smaller numbers, or not at all, in the other F fractions. It is suggested that these ultrastructurally distinct particles represent the biochemically distinct multiprotein complexes that constitute the cellulase-xylanase system of C. papyrosolvens C7. Some of the particles consisted of tightly packed globular components that appeared to be arranged in the shape of a ring with conical structures pointing out along its axis. Other particles had triangular, polyhedral, or star shapes. The major protein fraction (F4) almost exclusively contained particles consisting of loosely aggregated components, many of which appeared to be arranged along filamentous structures. The ultrastructural observations reported here support our previous conclusion that the cellulase-xylanase system of C. papyrosolvens C7 comprises at least seven different high-molecular-weight multiprotein complexes. Furthermore, results of this and earlier studies indicate that the interactions between C. papyrosolvens C7 and cellulose are different from those that have been described for Clostridium thermocellum.
Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor
Authors: Taguchi S , Suzuki M , Kojima S , Miura K , Momose H
Published: November 2016

Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'.
Isolation of the Anabaena sp. strain PCC 7120 sigA gene in a transcriptional-interference selection
Authors: Ramasubramanian T S , Pu F , Golden J W
Published: November 2016

A transcriptional-interference selection was performed to identify genes of Anabaena sp. strain PCC 7120 that encode DNA-binding proteins able to bind to the rbcL promoter. Unexpectedly, the selection yielded the previously identified sigA gene, which encodes the principal sigma factor. Protein extracts from Escherichia coli containing the sigA gene bound the rbcL promoter fragment in mobility shift assays, and competition experiments indicated binding to rbcL and glnA but not xisA or nifH upstream regions.
Subunit interactions and protein stability in the cyanobacterial light-harvesting proteins
Authors: Plank T , Toole C , Anderson L K
Published: November 2016

Strain 4R is a phycocyanin-minus mutant of the unicellular cyanobacterium Synechocystis sp. strain 6803. Although it lacks the light-harvesting protein phycocyanin, 4R has normal levels of phycocyanin (cpc) transcripts. Sequence analysis of the cpcB gene encoding the phycocyanin beta subunit shows an insertion mutation in 4R that causes early termination of translation. Other work has shown that the phycocyanin alpha subunit and the linker proteins encoded on the cpc transcripts are all functional in 4R, yet the defective phycocyanin beta subunit results in the complete absence of the alpha subunit and the linkers. Phycocyanin-minus mutants were constructed in a wild-type background by interruption of cpcB and cpcA with an antibiotic resistance gene and were compared with the 4R strain. Immunoblot analysis of the mutants demonstrated that interruption of one subunit was accompanied by a complete absence of the unassembled partner subunit. Phycocyanin assembly begins with the formation of the alpha beta heterodimer (the monomer) and continues through higher-order trimeric and hexameric aggregates that associate with linker proteins to form the phycobilisome rods. The results in this paper indicate that monomer formation is a critical stage in the biliprotein assembly pathway and that unassembled subunits are subject to stringent controls that prevent their appearance in vivo.
Heterologous assembly and rescue of stranded phycocyanin subunits by expression of a foreign cpcBA operon in Synechocystis sp. strain 6803
Authors: Plank T , Anderson L K
Published: November 2016

Light harvesting in cyanobacteria is performed by the biliproteins, which are organized into membrane-associated complexes called phycobilisomes. Most phycobilisomes have a core substructure that is composed of the allophycocyanin biliproteins and is energetically linked to chlorophyll in the photosynthetic membrane. Rod substructures are attached to the phycobilisome cores and contain phycocyanin and sometimes phycoerythrin. The different biliproteins have discrete absorbance and fluorescence maxima that overlap in an energy transfer pathway that terminates with chlorophyll. A phycocyanin-minus mutant in the cyanobacterium Synechocystis sp. strain 6803 (strain 4R) has been shown to have a nonsense mutation in the cpcB gene encoding the phycocyanin beta subunit. We have expressed a foreign phycocyanin operon from Synechocystis sp. strain 6701 in the 4R strain and complemented the phycocyanin-minus phenotype. Complementation occurs because the foreign phycocyanin alpha and beta subunits assemble with endogenous phycobilisome components. The phycocyanin alpha subunit that is normally absent in the 4R strain can be rescued by heterologous assembly as well. Expression of the Synechocystis sp. strain 6701 cpcBA operon in the wild-type Synechocystis sp. strain 6803 was also examined and showed that the foreign phycocyanin can compete with the endogenous protein for assembly into phycobilisomes.
Growth on octane alters the membrane lipid fatty acids of Pseudomonas oleovorans due to the induction of alkB and synthesis of octanol
Authors: Chen Q , Janssen D B , Witholt B
Published: November 2016

Growth of Pseudomonas oleovorans GPo1, which contains the OCT plasmid, on octane results in changes in the membrane phospholipid fatty acid composition. These changes were not found for GPo12, an OCT-plasmid-cured variant of GPo1, during growth in the presence or absence of octane, implying the involvement of OCT-plasmid-encoded functions. When recombinant strain GPo12(pGEc47) carrying the alk genes from the OCT plasmid was grown on octane, the cells showed the same changes in fatty acid composition as those found for GPo1, indicating that such changes result from induction and expression of the alk genes. This finding was corroborated by inducing GPo12(pGEc47) with dicyclopropylketone (DCPK), a gratuitous inducer of the alk genes. Further experiments showed that the increase of the mean acyl chain length of fatty acids is related to the expression of alkB, which encodes a major integral membrane protein, while the formation of trans unsaturated fatty acids mainly results from the effects of 1-octanol, an octane oxidation product.
Structure and organization of plasmid genes required to produce the translation inhibitor microcin C7
Authors: González-Pastor J E , San Millán J L , Castilla M A , Moreno F
Published: November 2016

The translation inhibitor microcin C7 (MccC7) is a linear heptapeptide whose N terminus has been replaced by an N-formyl group and whose C terminus has been replaced by the phosphodiester of 5'-adenylic acid and n-aminopropanol (J. I. Guijarro, J. E. González-Pastor, F. Baleux, J. L. San Millán, M. A. Castilla, M. Rico, F. Moreno, and M. Delepierre, J. Biol. Chem. 270:23520-23532, 1995). MccC7 production and immunity determinants lie on a 6.2-kb region of the Escherichia coli plasmid pMccC7. This region was entirely sequenced. It contains six open reading frames, which were shown to be true genes by different complementary approaches. Five genes, mccABCDE, which are transcribed in the same direction, are required to produce mature extracellular microcin. The sixth gene, mccF, adjacent to mccE, is transcribed in the opposite direction and encodes specific self-immunity. Genes mccA to -E constitute an operon transcribed from a promoter (mccp) located upstream of mccA. mccA is 21 nucleotides long and encodes the unmodified heptapeptide (J. E. González-Pastor, J. L. San Millán, and F. Moreno, Nature [London] 369:281, 1994). A comparison of predicted gene polypeptide products with those included in databases shows that an 81-amino-acid stretch of MccB is strikingly homologous to fragments of the same length of proteins ThiF and ChlN from E. coli, HesA from Anabaena sp. strain PCC7120, and UBA1, the ubiquitin-activating enzyme from different eukaryotic species. MccC displays several hydrophobic domains, suggesting a transmembrane location. The carboxyl end of MccE displays 41.2% identity with RimL, a protein required to acetylate the ribosome protein L12 from E. coli. In the absence of the other mcc genes, mccA impairs the growth of host cells, suggesting that unmodified MccA has antibiotic activity. A model for MccC7 biosynthesis, export, and immunity is proposed.
Purification of the copper response extracellular proteins secreted by the copper-resistant methanogen Methanobacterium bryantii BKYH and cloning, sequencing, and transcription of the gene encoding these proteins
Authors: Kim B K , Pihl T D , Reeve J N , Daniels L
Published: November 2016

When the copper-resistant methanogen Methanobacterium bryantii BKYH was exposed to 1 mM Cu(II), it secreted approximately fourfold increased levels of three proteins, copper response extracellular (CRX) proteins. The members of the CRX protein trio had apparent molecular masses of 40.8, 42.3, and 42.9 kDa and were purified together from the culture supernatant and separated from each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the three proteins were essentially identical, and antibodies raised against one of the trio reacted with all three proteins and with three other intracellular proteins with slightly higher molecular weights. The N-terminal amino acid sequence of one of these larger proteins was different from that of the secreted CRX proteins. The gene crx, which encodes the CRX proteins, was cloned and sequenced, and crx transcription was characterized. The crx sequence predicts that the encoded polypeptide is synthesized as a precursor with an N-terminal leader peptide, containing 28 amino acid residues, that is removed during the extracellular secretion of the CRX proteins. Transcription was initiated 274 bp upstream from the crx gene, producing an approximately 1.4-kb monocistronic transcript that was present in M. bryantii BKYH cells under all growth conditions but that increased approximately fourfold in vivo in response to Cu addition. The CRX proteins appear to be glycosylated, since they react with concanavalin A and neuraminidase, and to be the products of one gene that have different levels of posttranslational glycosylation. This is supported by very similar chromatographic and electrophoretic properties, identical N-terminal amino acid sequences, immunological cross-reactivities, and the detection of only one crx-related sequence by Southern blotting. Western blots (immunoblots) showed no evidence for CRX proteins in cell lysates of several other Methanobacterium strains.
Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis
Authors: McDonough M , Sangan P , Gonda D K
Published: November 2016

Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiquitin-conjugating (E2) enzyme is required for cellular processes as diverse as DNA repair, selective proteolysis, and normal growth. For most RAD6-dependent functions, the relevant in vivo targets, as well as the mechanisms and cofactors that govern RAD6 substrate selectivity, are unknown. We have explored the utility of "charge-to-alanine" scanning mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that are nevertheless severely handicapped with respect to several in vivo functions. Five of the nine mutants we generated show defects in their in vivo functions, but almost all of the most severely affected mutants displayed unfacilitated ubiquitin-conjugating activity in vitro. We suggest that E2 mutants obtained by this approach are likely to be defective with respect to interaction with other, trans-acting factors required for their intracellular activity or substrate selectivity and therefore will be useful for further genetic and biochemical studies of ubiquitin-conjugating enzyme function.
Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration
Authors: Hochkoeppler A , Jenney F E , Lang S E , Zannoni D , Daldal F
Published: November 2016

We have recently established that the facultative phototrophic bacterium Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center during photosynthesis (F.E. Jenney and F. Daldal, EMBO J. 12:1283-1292, 1993; F.E. Jenney, R.C. Prince, and F. Daldal, Biochemistry 33:2496-2502, 1994). One pathway is via the well-characterized, water-soluble cytochrome c2 (cyt c2), and the other is via a novel membrane-associated c-type cytochrome named cyt cy. In this work, we probed the role of cyt cy in respiratory electron transport by isolating a set of R. capsulatus mutants lacking either cyt c2 or cyt cy, in the presence or in the absence of a functional quinol oxidase-dependent alternate respiratory pathway. The growth and inhibitor sensitivity patterns of these mutants, their respiratory rates in the presence of specific inhibitors, and the oxidation-reduction kinetics of c-type cytochromes monitored under appropriate conditions demonstrated that cyt cy, like cyt c2, connects the bc1 complex and the cyt c oxidase during respiratory electron transport. Whether cyt c2 and cyt cy are the only electron carriers between these two energy-transducing membrane complexes of R. capsulatus is unknown.
Very short patch repair of T:G mismatches in vivo: importance of context and accessory proteins
Authors: Lieb M , Rehmat S
Published: November 2016

In Escherichia coli, T:G mismatches in specific contexts are corrected by a very short patch (VSP) repair system. Previous studies have shown that the product of gene vsr mediates correction of T:G to C:G in the 5'CTAGG/3'GGTCC context and in some related contexts. Amber mutations that arose in CAG sequences in gene cI of bacteriophage lambda were used to determine the effect of flanking bases on the repair of T:G mispairs arising during phage recombination. The experimental findings were combined with published data on mismatch repair of mutations in lambda gene P and E. coli gene lacI. While VSP repair was most efficient in the context 5'CTAGG, there was very significant correction when either the 5'C or the 3' G was replaced by another base. Some mismatch repair of TAG to CAG occurred in all contexts tested. Reduction in VSP repair caused by the lack of MutL or MutS was fully complemented by the addition of vsr+ plasmids when the T:G mispair was in the 5'CTAGG/3'GGTCC context. VSP repair was decreased in bacteria containing mutS+ on a multicopy plasmid. It is suggested that VSP repair maintains sequences such as the repetitive extragenic palindromic (REP) and Chi sequences, which have important roles in E. coli and closely related bacteria.