bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile
Authors: Erauso G , Marsin S , Benbouzid-Rollet N , Baucher M F , Barbeyron T , Zivanovic Y , Prieur D , Forterre P
Published: November 2016

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.
Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor
Authors: Kim K S , Farrand S K
Published: November 2016

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.
Cyclic AMP receptor protein positively controls gyrA transcription and alters DNA topology after nutritional upshift in Escherichia coli
Authors: Gomez-Gomez J M , Baquero F , Blazquez J
Published: November 2016

The expression of a transcriptional gyrA-lacZ gene fusion throughout the Escherichia coli growth cycle and the effect that mutation delta crp39 had on this expression were studied. The data obtained indicate that the expression of gyrA is growth phase dependent and under the positive control of the cyclic AMP receptor protein (CRP). Complementation analysis of gyrA-lacZ expression with wild-type CRP or variant CRP pc (with a T-to-A mutation at position 158) in a CRP-deficient background suggests that this CRP action is mediated by a class I or class II CRP-dependent promoter(s). Our results also indicate that CRP may be involved in the modulation of DNA topology in the transition from the lag period to the exponential phase of growth.
Genes associated with meningococcal capsule complex are also found in Neisseria gonorrhoeae
Authors: Petering H , Hammerschmidt S , Frosch M , van Putten J P , Ison C A , Robertson B D
Published: November 2016

A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.
Analysis of suppressor mutations of spoIVCA mutations: occurrence of DNA rearrangement in the absence of site-specific DNA recombinase SpoIVCA in Bacillus subtilis
Authors: Sato T , Harada K , Kobayashi Y
Published: November 2016

The spoIVCA gene of Bacillus subtilis encodes a site-specific recombinase, which excises a 48-kb skin element from the chromosomal DNA by DNA rearrangement and creates a new composite gene, sigK, on the chromosome. From spoIVCA mutants, we have isolated Spo+ revertants which have no skin element but have an intact sigK gene. This result suggests that the DNA rearrangement can occur in the absence of spoIVCA.
redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD
Authors: Fujii T , Gramajo H C , Takano E , Bibb M J
Published: November 2016

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first experimental evidence that HrdD can function as a sigma factor.
High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis
Authors: Xavier K B , Martins L O , Peist R , Kossmann M , Boos W , Santos H
Published: November 2016

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.
Cloning of a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH): evidence for a second meningococcal L-LDH with different regulation
Authors: Erwin A L , Gotschlich E C
Published: November 2016

We report the cloning of lldA, a Neisseria meningitidis gene for L-lactate dehydrogenase (L-LDH). Escherichia coli contains a single L-LDH gene (lldD) in the lld operon (previously lct). E. coli grown in complex media does not have L-LDH activity, but the activity is induced by growth in defined medium with L-lactate as the carbon source. In contrast, meningococci contain at least one L-LDH in addition to the lldA gene product. These enzymes are active in meningococci grown in complex media and are not dependent on growth in L-lactate. The predicted amino acid sequence of lldA is homologous to that of E. coli lldD and of other prokaryotic and eukaryotic flavin mononucleotide-containing enzymes that catalyze the oxidation of L-lactate and other small alpha-hydroxy acids. A mutant with a deletion in lldA was found to have reduced L-LDH activity. However, this mutant was able to grow on L-lactate, indicating that a second L-LDH must exist. Activity of the lldA enzyme was affected by growth conditions, being increased by growth on a defined medium with either L-lactate or pyruvate as the carbon source. For meningococci grown on a complex medium, activity of the lldA enzyme was increased by growth on plates or in well-aerated broth. A second L-lactate-oxidizing activity was seen in bacteria grown in poorly aerated broth. Neisseria gonorrhoeae contains a homolog of lldA. As for meningococci, mutation of the gonococcal lldA reduced L-LDH activity but did not affect growth on L-lactate.
The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942
Authors: Eriksson M J , Clarke A K
Published: November 2016

The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.
A study of mycobacterial transcriptional apparatus: identification of novel features in promoter elements
Authors: Bashyam M D , Kaushal D , Dasgupta S K , Tyagi A K
Published: November 2016

Our earlier studies on transcriptional signals of mycobacteria had revealed that (i) strong promoters occur less frequently in the slowly growing pathogen Mycobacterium tuberculosis H37Rv than in the fast-growing saprophyte M. smegmatis and (ii) mycobacterial promoters function poorly in Escherichia coli. We now present evidence that RNA polymerases of M. smegmatis, M. tuberculosis, and M. bovis BCG recognize promoter elements with comparable efficiencies. Analysis of these randomly isolated mycobacterial promoters by DNA sequencing, primer extension, and deletion experiments revealed that their -10 regions are highly similar to those of E. coli promoters, in contrast to their -35 regions, which can tolerate a greater variety of sequences, owing presumably to the presence of multiple sigma factors with different or overlapping specificities for -35 regions, as reported earlier for the Streptomyces promoters. A comparison of the -10 and -35 binding domains of MysA, HrdB, and RpoD (the principal sigma factors of M. smegmatis, Streptomyces aureofaciens, and E. coli, respectively) showed that all three sigma factors have nearly identical -10 binding domains. However, the -35 binding domains of the principal mycobacterial and streptomycete sigma factors, although nearly identical to each other, are vastly different from the corresponding region of the sigma factor of E. coli. Thus, the transcriptional signals of mycobacteria have features in common with Streptomyces promoters but differ from those of E. coli because of major differences in the -35 regions of the promoters and the corresponding binding domain in the sigma factor.
Identification and characterization of iron-regulated Bordetella pertussis alcaligin siderophore biosynthesis genes
Authors: Kang H Y , Brickman T J , Beaumont F C , Armstrong S K
Published: November 2016

Bordetella bronchiseptica mutants BRM1, BRM6, and BRM9 fail to produce the native dihydroxamate siderophore alcaligin. A 4.5-kb BamHI-Smal Bordetella pertussis genomic DNA fragment carried multiple genes required to restore alcaligin production to these siderophore-deficient mutants. Phenotypic complementation analysis using subclones of the 4.5-kb genomic region demonstrated that the closely linked BRM1 and BRM9 mutations were genetically separable from the BRM6 mutation, and both insertions exerted strong polar effects on expression of the downstream gene defined by the BRM6 mutation, suggesting a polycistronic transcriptional organization of these alcaligin biosynthesis genes. Subcloning and complementation experiments localized the putative Bordetella promoter to a 0.7-kb BamHI-SphI subregion of the cloned genomic DNA fragment. Nucleotide sequencing, phenotypic analysis of mutants, and protein expression by the 4.5-kb DNA fragment in Escherichia coli suggested the presence of three alcaligin system genes, namely, alcA, alcB, and alcC. The deduced protein products of alcA, alcB, and alcC have significant primary amino acid sequence similarities with known microbial siderophore biosynthesis enzymes. Primer extension analysis mapped the transcriptional start site of the putative alcaligin biosynthesis operon containing alcABC to a promoter region overlapping a proposed Fur repressor-binding site and demonstrated iron regulation at the transcriptional level.
Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid
Authors: Stevenson G , Andrianopoulos K , Hobbs M , Reeves P R
Published: November 2016

Colanic acid (CA) is an extracellular polysaccharide produced by most Escherichia coli strains as well as by other species of the family Enterobacteriaceae. We have determined the sequence of a 23-kb segment of the E. coli K-12 chromosome which includes the cluster of genes necessary for production of CA. The CA cluster comprises 19 genes. Two other sequenced genes (orf1.3 and galF), which are situated between the CA cluster and the O-antigen cluster, were shown to be unnecessary for CA production. The CA cluster includes genes for synthesis of GDP-L-fucose, one of the precursors of CA, and the gene for one of the enzymes in this pathway (GDP-D-mannose 4,6-dehydratase) was identified by biochemical assay. Six of the inferred proteins show sequence similarity to glycosyl transferases, and two others have sequence similarity to acetyl transferases. Another gene (wzx) is predicted to encode a protein with multiple transmembrane segments and may function in export of the CA repeat unit from the cytoplasm into the periplasm in a process analogous to O-unit export. The first three genes of the cluster are predicted to encode an outer membrane lipoprotein, a phosphatase, and an inner membrane protein with an ATP-binding domain. Since homologs of these genes are found in other extracellular polysaccharide gene clusters, they may have a common function, such as export of polysaccharide from the cell.
Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA
Authors: Xie Z D , Hershberger C D , Shankar S , Ye R W , Chakrabarty A M
Published: November 2016

Conversion from the nonmucoid to the mucoid phenotype is a typical feature of Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients. One of the key genetic controls in this conversion to mucoidy is from the algT(U)-mucA-mucB(algN) locus, located at 67.5 min on the standard P. aeruginosa chromosomal map. The algT gene promotes conversion to mucoidy and encodes an alternative sigma factor (sigma E) which belongs to the ECF (for extracytoplasmic function) family. On the other hand, the mucA and mucB (algN) genes suppress conversion to mucoidy. Loss-of-function mutations in mucA have been postulated to be the cause of mucoidy in some P. aeruginosa strains isolated from cystic fibrosis patients. We expressed and purified the protein products from the mucA and mucB open reading frames. The purified MucA protein abolishes the in vitro transcription specified by AlgT and the ability of AlgT to compete with an Escherichia coli sigma factor, FliA, suggesting that inhibiting AlgT-dependent transcription could be the mechanism by which mucA suppresses mucoidy in vivo. Enzyme-linked immunosorbent assay and glycerol density gradient sedimentation experiments suggest that MucA physically interacts with AlgT.
asmB, a suppressor locus for assembly-defective OmpF mutants of Escherichia coli, is allelic to envA (lpxC)
Authors: Kloser A W , Laird M W , Misra R
Published: November 2016

A novel genetic scheme allowed us to isolate extragenic suppressor mutations that restored mutant OmpF assembly. One group of these mutations, termed asmB for assembly suppressor mutation B, permitted mutant OmpF assembly in a non-allele-specific manner. Genetic mapping analyses placed the asmB mutations at the 2-min region of the Escherichia coli K-12 chromosome. Further analyses revealed that the asmB mutations map within the envA (lpxC) gene, which encodes an enzyme needed for the synthesis of the lipid A moiety of lipopolysaccharide (LPS). Nucleotide sequence analysis showed that the asmB mutations caused a change from F-50 to S (F50S substitution) (asmB2 and asmB3) or a G210S substitution (asmB1) in EnvA. Cells bearing the asmB alleles displayed increased sensitivity to various hydrophobic compounds and detergents, suggesting an alteration within the outer membrane. Direct examination (of the LPS showed that its amounts were reduced by the asmB mutations, with asmB1 exerting a greater effect than asmB2 or asmB3. Thus, it appears that the asmB mutations achieve mutant OmpF assembly suppression by reducing LPS levels, which in turn may alter membrane fluidity.
Identification of a Cryptococcus neoformans gene that directs expression of the cryptic Saccharomyces cerevisiae mannitol dehydrogenase gene
Authors: Perfect J R , Rude T H , Wong B , Flynn T , Chaturvedi V , Niehaus W
Published: November 2016

The Mtl gene from Cryptococcus neoformans, which confers the ability of Saccharomyces cerevisiae Sc4l YJO to grow on mannitol with substantial NAD-dependent mannitol dehydrogenase activity, was identified. Purifications and characterizations of this enzyme show that it is found in polyploid strain BB1, and the peptide sequence of the enzyme helped identify the saccharomyces gene encoding this mannitol dehydrogenase activity. On the other hand, the Mtl gene of C. neoformans encodes a 346-amino-acid protein which is not mannitol dehydrogenase but a regulatory element which is active in a heterologous fungus.
Evidence that the PBP 5 synthesis repressor (psr) of Enterococcus hirae is also involved in the regulation of cell wall composition and other cell wall-related properties
Authors: Massidda O , Kariyama R , Daneo-Moore L , Shockman G D
Published: November 2016

psr has been reported by M. Ligozzi, F. Pittaluga, and R. Fontana, (J. Bacteriol. 175:2046-2051, 1993) to be a genetic element located just upstream of the structural gene for the low-affinity penicillin-binding protein 5 (PBP 5) in the chromosome of Enterococcus hirae ATCC 9790 and to be involved in the repression of PBP 5 synthesis. By comparing properties of strains of E. hirae that contain a full-length, functional psr with those of strains that possess a truncated form of the gene, we have obtained data that indicate that psr is involved in the regulation of several additional surface-related properties. We observed that cells of strains that possessed a truncated psr were more sensitive to lysozyme-catalyzed protoplast formation, autolyzed more rapidly in 10 mM sodium phosphate (pH 6.8), and, in contrast to strains that possess a functional psr, retained these characteristics after the cultures entered the stationary growth phase. Cellular lytic properties did not correlate with differences in the cellular contents of muramidase-1 or muramidase-2, with the levels of PBP 5 produced, or with the penicillin susceptibilities of the strains. However, a strong correlation was observed with the amounts of rhamnose present in the cell walls of the various strains. All of the strains examined that possessed a truncated form of psr also possessed approximately one-half of the rhamnose content present in the walls of strains that possessed a functional psr. These data suggest that psr is also involved in the regulation of the synthesis of, or covalent linkage to the cell wall peptidoglycan of, a rhamnose-rich polysaccharide. These differences in cell wall composition could be responsible for the observed phenotypic differences. However, the multiple effects of psr suggest that it is part of a global regulatory system that, perhaps independently, affects several cell surface-related properties.
Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2)
Authors: McCormick J R , Losick R
Published: November 2016

We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.
Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens
Authors: Matthysse A G , Yarnall H A , Young N
Published: November 2016

Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously. A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified. Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange. The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells. We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH). The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria. The homology was strongest to proteins involved in the transport of spermidine and putrescine. The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A. tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni. The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells.
Absence of light-induced proton extrusion in a cotA-less mutant of Synechocystis sp. strain PCC6803
Authors: Katoh A , Sonoda M , Katoh H , Ogawa T
Published: November 2016

cotA of Synechocystis sp. strain PCC6803 was isolated as a gene that complemented a mutant defective in CO2 transport and is homologous to cemA that encodes a chloroplast envelope membrane protein (A. Katoh, K.S. Lee, H. Fukuzawa, K. Ohyama, and T. Ogawa, Proc. Natl. Acad. Sci. USA 93:4006-4010, 1996). A mutant (M29) constructed by replacing cotA in the wild-type (WT) Synechocystis strain with the omega fragment was unable to grow in BG11 medium (approximately 17 mM Na+) at pH 6.4 or at any pH in a low-sodium medium (100 microM Na+) under aeration with 3% (vol/vol) CO2 in air. The WT cells grew well in the pH range between 6.4 and 8.5 in BG11 medium but only at alkaline pH in the low-sodium medium. Illumination of the WT cells resulted in an extrusion followed by an uptake of protons. In contrast, only proton uptake was observed for the M29 mutant in the light without proton extrusion. There was no difference in sodium uptake activity between the WT and mutant. The mutant still possessed 51% of the WT CO2 transport activity in the presence of 15 mM NaCl. On the basis of these results we concluded that cotA has a role in light-induced proton extrusion and that the inhibition of CO2 transport in the M29 mutant is a secondary effect of the inhibition of proton extrusion.
Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12
Authors: Poole R K , Anjum M F , Membrillo-Hernández J , Kim S O , Hughes M N , Stewart V
Published: November 2016

Escherichia coli possesses a soluble flavohemoglobin, with an unknown function, encoded by the hmp gene. A monolysogen containing an hmp-lacZ operon fusion was constructed to determine how the hmp promoter is regulated in response to heme ligands (O2, NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite). Expression of the phi (hmp-lacZ)1 fusion was similar during aerobic growth in minimal medium containing glucose, glycerol, maltose, or sorbitol as a carbon source. Mutations in cya (encoding adenylate cyclase) or changes in medium pH between 5 and 9 were without effect on aerobic expression. Levels of aerobic and anaerobic expression in glucose-containing minimal media were similar; both were unaffected by an arcA mutation. Anaerobic, but not aerobic, expression of phi (hmp-lacZ)1 was stimulated three- to four-fold by an fnr mutation; an apparent Fnr-binding site is present in the hmp promoter. Iron depletion of rich broth medium by the chelator 2'2'-dipyridyl (0.1 mM) enhanced hmp expression 40-fold under anaerobic conditions, tentatively attributed to effects on Fnr. At a higher chelator concentration (0.4 mM), hmp expression was also stimulated aerobically. Anaerobic expression was stimulated 6-fold by the presence of nitrate and 25-fold by the presence of nitrite. Induction by nitrate or nitrite was unaffected by narL and/or narP mutations, demonstrating regulation of hmp by these ions via mechanisms alternative to those implicated in the regulation of other respiratory genes. Nitric oxide (10 to 20 microM) stimulated aerobic phi (hmp-lacZ)1 activity by up to 19-fold; soxS and soxR mutations only slightly reduced the NO effect. We conclude that hmp expression is negatively regulated by Fnr under anaerobic conditions and that additional regulatory mechanisms are involved in the responses to oxygen, nitrogen compounds, and iron availability. Hmp is implicated in reactions with small nitrogen compounds.
Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes
Authors: Chen Q , Janssen D B , Witholt B
Published: November 2016

The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS. Growth of the wild-type strain GPo1 and P. oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology. These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes. These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons. Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes. Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes. This resulted in a return of the recombinants to normal physiology and growth.
Development of a temperature-inducible expression system for Streptomyces spp
Authors: Kataoka M , Tatsuta T , Suzuki I , Kosono S , Seki T , Yoshida T
Published: November 2016

PCR mutagenesis of a 0.9-kbp fragment, containing a repressor gene, traR, and its target promoter, Ptra, from Streptomyces nigrifaciens plasmid pSN22, produced Streptomyces lividans clones with temperature-inducible Ptra expression. Using the promoterless gene for the thermostable Thermus flavus malate dehydrogenase as an indicator, an induction of enzyme activity of as much as was observed in a temperature shift from 28 to 37 degrees C. Temperature downshift reestablished repression of Ptra, making these promoter cassettes very attractive for the temporally regulated expression of cloned genes in Streptomyces spp.
Mutation and Mutagenesis of thiol peroxidase of Escherichia coli and a new type of thiol peroxidase family
Authors: Cha M K , Kim H K , Kim I H
Published: November 2016

A novel thioredoxin-linked thiol peroxidase (Px) from Escherichia coli has been reported previously (M. K. Cha, H. K. Kim, and I. H. Kim, J. Biol. Chem. 270:28635-28641, 1995). In an attempt to perform physiological and biochemical characterizations of the thiol Px, a thiol Px null (tpx) mutant and a functional-residue mutant of thiol Px were produced. The tpx mutant was viable in aerobic culture but grew more slowly than the wild-type cells. The difference in growth rate became more pronounced when oxidative-stress-inducing reagents, such as peroxides and paraquat, were added to the cultures. The viability of the individual tpx mutant under oxidative stress was much lower than that of wild-type cells. tpx mutants growing aerobically respond to paraquat with a sixfold greater induction of Mn-superoxide dismutase than that of the wild-type cells. The deduced amino acid sequence of the thiol Px was found to be from 42 to 72% identical to the sequences of proteins from Haemophilus influenzae (ToxR regulon), Vibrio cholerae (ToxR regulon), and three kinds of streptococci (coaggregation-mediating adhesins), suggesting that they all belong to a new thiol Px family. Alignment of the amino acid sequences of the thiol Px family members showed that one cysteine, which corresponds to Cys-94 in E. coli thiol Px, is perfectly conserved. The substitution of serine for this cysteine residue resulted in complete loss of Px activity. These results suggest that the members of the thiol Px family, including E. coli thiol Px, have a functional cysteine residue and function in vivo as peroxidases.
A novel developmental stage-specific lectin of the basidiomycete Pleurotus cornucopiae
Authors: Oguri S , Ando A , Nagata Y
Published: November 2016

A novel lectin was isolated from mycelia of the basidiomycete Pleurotus cornucopiae grown on solid medium. The lectin was purified to homogeneity by mucin-Sepharose affinity chromatography. The molecular mass of the lectin was 40 kDa under reducing conditions, but the subunits were polymerized through disulfide bridges under physiological conditions. Hemagglutinating activity of this lectin was completely inhibited by 2-mercaptoethanol, indicating that the multimer is active. The activity was also inhibited by EDTA, and restored by CaCl2. N-Acetyl-D-galactosamine was the most potent hapten inhibitor. N-terminal amino acid sequence analysis revealed that the mycelial lectin was different from the fruit body lectin of this organism. The mycelial lectin appeared prior to fruit body formation and disappeared during the formation of fruit bodies. The lectin was localized on the surface of solid-medium-grown mycelia, and only dikaryotic, and not monokaryotic, mycelia produced the lectin. These results suggest that the appearance of this lectin is associated with fruit body formation.
Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp. strain IGTS8 as a sulfide/sulfoxide monooxygenase
Authors: Lei B , Tu S C
Published: November 2016

The oxidation of dibenzothiophene to dibenzothiophene sulfone has been linked to the enzyme encoded by the sox/dszC gene from Rhodococcus sp. strain IGTS8 (S. A. Denome, C. Oldfield, L. J. Nash, and K. D. Young, J. Bacteriol. 176:6707-6717, 1994; C. S. Piddington, B. R. Kovacevich, and J. Rambosek, Appl. Environ. Microbiol. 61:468-475, 1995). However, this enzyme has not been characterized, and the type of its catalytic activity remains unclassified. In this work, the sox/dszC gene was overexpressed in Escherichia coli, a procedure for the purification of the expressed enzyme was developed, and the properties of and the reactions catalyzed by the purified enzyme were characterized. This enzyme binds one flavin mononucleotide (Kd, 7 micrometers) or reduced flavin mononucleotide (FMNH2) (Kd < 10(-8) M) per 90,200-Da homodimer, and FMNH2 is an essential cosubstrate for its activity. Patterns of product formation were examined under different FMNH2 availabilities, and results indicate that this enzyme catalyzes a stepwise conversion of dibenzothiophene to the corresponding sulfoxide and subsequently to the sulfone. On the basis of isotope labeling patterns with H2(18)O and 18O2, dibenzothiophene sulfoxide and sulfone obtained their oxygen atom(s) from molecular oxygen rather than water in their formation from dibenzothiophene. The enzyme also utilizes benzyl sulfide and benzyl sulfoxide as substrates. Hence, it is identified as a sulfide/sulfoxide monooxygenase. This monooxygenase is similar to the microsomal flavin-containing monooxygenase but is unique among microbial flavomonooxygenases in its ability to catalyze two consecutive monooxygenation reactions.
Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock
Authors: Tsui H C , Feng G , Winkler M E
Published: November 2016

The amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of Escherichia coli contains genes that are important for diverse cellular functions, including DNA mismatch repair (mutL), tRNA modification (miaA), pleiotropic regulation (hfq), and proteolysis (hflX-hflK-hflC). We show that this superoperon contains three E simga(32)-dependent heat shock promoters, P(mutL)HS,P(miaA)HS, and P1(hfq)HS, in addition to four E sigma(70)-dependent promoters, P(mutL), P(miaA), P2(hfq), and P3(hfq). Transcripts from P(mutL)HS and P(miaA)HS were most prominent in vivo during extreme heat shock (50 degrees C), whereas P1(hfq)HS transcripts were detectable under nonshock conditions and increased significantly after heat shock at 50 degrees C. The P(mutL)HS, P(miaA)HS, and P1(hfq)HS transcripts were not detected in an rpoH null mutant. All three promoters were transcribed by E sigma (32) in vitro at 37 degrees C and contain -35 and -10 regions that resemble the E sigma(32) consensus. In experiments to assess the possible physiological relevance of the P(mutL)HS and P(miaA)HS promoters, we found that E. coli prototrophic strain MG 1655 increased in cell mass and remained nearly 100% viable for several hours at 50 degrees C in enriched media. In these cells, a significant fraction of mutL and hfq-hflA region transcripts were from P(mutL)HS and P1(hfq)HS, respectively, and the amounts of the miaA, hfq, hflX, hflK, and hflC transcripts increased in comparison with those in nonstressed cells. The cellular amounts of MutL and the hfq gene product (HF-I protein) were maintained during heat shock at 44 or 50 degrees C. Consistent with their expression patterns, miaA and hfq were essential for growth and viability, respectively, at temperatures of 45 degrees C and above. Together, these results suggest that there is a class of E sigma(32) promoters that functions mainly at high temperatures to ensure E. coli function and survival.
Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome
Authors: Ahsan M M , Kimura T , Karita S , Sakka K , Ohmiya K
Published: November 2016

The Clostridium thermocellum F1 celJ gene, encoding endoglucanase J (CelJ), consists of an open reading frame (ORF) of 4,803 nucleotides and encodes a protein of 1,601 amino acids with a molecular weight of 178,055. The ORF was confirmed as celJ by comparison with the N-terminal sequence of a truncated CelJ derivative. CelJ is a modular enzyme composed of N-terminal signal peptide and six domains in the following order: an S-layer homology domain, a domain of unknown function (UD-1), a subfamily E1 endoglucanase domain, a family J endoglucanase domain, a docking domain, and another domain of unknown function (UD-2). UD-1 has no significant similarity to UD-2. CelJ hydrolyzed carboxymethylcellulose and xylan, and xylanase activity was ascribed to the family J domain. Antiserum raised against the truncated CelJ cross-reacted with proteins contained in the cellulosome of C. thermocellum F1. These results strongly suggest that CelJ is equivalent to S2, which was identified as the largest catalytic component in the cellulosome of C. thermocellum YS. A second but incomplete ORF encoding an enzyme classified in subfamily E2 endoglucanase, was located downstream of celJ.
Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis
Authors: Guasch J F , Piqué N , Climent N , Ferrer S , Merino S , Rubires X , Tomas J M , Regué M
Published: November 2016

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.
Molecular, genetic, and biochemical characterization of the serC gene of Methanosarcina barkeri Fusaro
Authors: Metcalf W W , Zhang J K , Shi X , Wolfe R S
Published: November 2016

The Methanosarcina barkeri serC gene, encoding phosphoserine aminotransferase, was cloned by complementation of an Escherichia coli serC mutant, and its nucleotide sequence was determined. The M. barkeri SerC protein shares significant homology with other known SerC proteins. E. coli serC hosts carrying the cloned gene express phosphoserine aminotransferase activity, verifying the function of this gene.
The cbpA chaperone gene function compensates for dnaJ in lambda plasmid replication during amino acid starvation of Escherichia coli
Authors: Wegrzyn A , Taylor K , Wegrzyn G
Published: November 2016

We found previously that lambda plasmid DNA replication in amino acid-starved Escherichia coli relA mutants (i.e., during the relaxed response), which is carried out by the inherited replication complex, is dependent on functions of DnaK and GrpE molecular chaperones but proceeds in a dnaj mutant at a nonpermissive temperature. Here we demonstrate that this replication is inhibited when functions of both dnaJ and cbpA are impaired. In complete media, the growth of the lambda pi A66 phage (capable of replicating in E. coli dnaJ, dnaK, and grpE missense mutants at 30 degrees C), as well as efficiency of transformation by the lambda pi A66 plasmid, is significantly decreased in a dnaJ259 cbpA::kan double mutant. These results strengthen the proposal of other authors (C. Ueguchi, M. Kakeda, H. Yamada, and T. Mizuno, Proc. Natl. Acad. Sci. USA 91:1054-1058, 1994; C. Ueguchi, T. Shiozawa, M. Kakeda, H. Yamada, and T. Mizuno, J. Bacteriol. 177:3894-3896, 1995; and T. Yamashino, M. Kakeda, C. Ueguchi, and T. Mizuno, Mol. Microbiol. 13:475-483, 1994) that the cbpA gene product is a functional analog of the DnaJ chaperone in E. coli.
The mitogen-activated protein kinase homolog HOG1 gene controls glycerol accumulation in the pathogenic fungus Candida albicans
Authors: San José C , Monge R A , Pérez-Díaz R , Pla J , Nombela C
Published: November 2016

The Candida albicans HOG1 gene (HOG1CA) was cloned by functional complementation of the osmosensitive phenotype associated with Saccharomyces cerevisiae hog1 delta mutants. HOG1CA codes for a 377-amino-acid protein, 78% identical to S. cerevisiae Hog1p. A C. albicans hog1 null mutant was found to be sensitive to osmotic stress and failed to accumulate glycerol on high-osmolarity media.
Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope
Authors: Pfanzagl B , Zenker A , Pittenauer E , Allmaier G , Martinez-Torrecuadrada J , Schmid E R , De Pedro M A , Löffelhardt W
Published: November 2016

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing.
Identification of a region of genetic variability among Bacillus anthracis strains and related species
Authors: Andersen G L , Simchock J M , Wilson K H
Published: November 2016

The identification of a region of sequence variability among individual isolates of Bacillus anthracis as well as the two closely related species, Bacillus cereus and Bacillus mycoides, has made a sequence-based approach for the rapid differentiation among members of this group possible. We have identified this region of sequence divergence by comparison of arbitrarily primed (AP)-PCR "fingerprints" generated by an M13 bacteriophage-derived primer and sequencing the respective forms of the only polymorphic fragment observed. The 1,480-bp fragment derived from genomic DNA of the Sterne strain of B. anthracis contained four consecutive repeats of CAATATCAACAA. The same fragment from the Vollum strain was identical except that two of these repeats were deleted. The Ames strain of B. anthracis differed from the Sterne strain by a single-nucleotide deletion. More than 150 nucleotide differences separated B. cereus and B. mycoides from B. anthracis in pairwise comparisons. The nucleotide sequence of the variable fragment from each species contained one complete open reading frame (ORF) (designated vrrA, for variable region with repetitive sequence), encoding a potential 30-kDa protein located between the carboxy terminus of an upstream ORF (designated orf1) and the amino terminus of a downstream ORF (designated lytB). The sequence variation was primarily in vrrA, which was glutamine- and proline-rich (30% of total) and contained repetitive regions. A large proportion of the nucleotide substitutions between species were synonymous. vrrA has 35% identity with the microfilarial sheath protein shp2 of the parasitic worm Litomosoides carinii.