bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and germination defects of Bacillus subtilis spores with altered contents of a spore coat protein
Authors: Bourne N , FitzJames P C , Aronson A I
Published: November 2016

The start sites for transcription and translation of a Bacillus subtilis spore coat protein gene, cotT, were determined. The CotT protein was synthesized as a 10.1-kDa precursor which was processed to a coat polypeptide of 7.8 kDa. Insertional inactivation of the cotT gene resulted in spores with an altered appearance of the inner coat layers and slow germination in response to a germination solution containing fructose, glucose, and asparagine. Rates of germination in L-alanine and in Penassay broth were the same as that of the wild type. A strain containing the cotT gene on a low-copy-number plasmid produced spores with an excess of CotT precursor and a thickening of the striated inner coat. These spores responded poorly to all of the germinants mentioned above. A site-directed mutation of the codon at the processing site of CotT resulted in the accumulation of the precursor in sporulating cells and on the spores, but there was no effect on the germination rates or solvent resistance of these spores. Both the lack and the overproduction of CotT led to subtle alterations in the structure of the inner spore coat and in the capacity of spores to respond to particular germinants.
Klebsiella aerogenes catabolite gene activator protein and the gene encoding it (crp)
Authors: Osuna R , Bender R A
Published: November 2016

The catabolite gene activator protein from Klebsiella aerogenes (CAPK) and the corresponding protein from Escherichia coli (CAPE) were shown to be nearly identical. Both CAPK and CAPE activated transcription from the CAP-dependent promoters derived from E. coli and K. aerogenes. The crp gene from K. aerogenes (encoding CAP) is tightly linked to rpsL. The nucleotide sequence of crp predicts an amino acid sequence for CAPK that differs in only one position from that of CAPE.
rRNA transcription rate in Escherichia coli
Authors: Gotta S L , Miller O L , French S L
Published: November 2016

The rate of in vivo transcription elongation for Escherichia coli rRNA operons was determined by electron microscopy following addition of rifampin to log-phase cultures. Direct observation of RNA polymerase positions along rRNA operons 30, 40, and 70 s after inhibition of transcription initiation yielded a transcription elongation rate of 42 nucleotides per s.
Expression of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria is not under the control of hrp genes and is independent of plant factors
Authors: Knoop V , Staskawicz B , Bonas U
Published: November 2016

The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria pepper race 1 is responsible for the induction of a race-specific hypersensitive reaction in resistant pepper cultivars. A DNA region of 3.7 kb, containing several open reading frames and an internal repetitive region, was shown previously to be necessary for avirulence activity (U. Bonas, R. E. Stall, and B. Staskawicz, Mol. Gen. Genet. 218:127-136, 1989). The promoter of avrBs3 was identified by using gene fusions to beta-glucuronidase. Also, we mapped the transcription start site and showed that the avrBs3 gene is expressed constitutively in cells grown in minimal or complex medium and in planta. Polyclonal antibodies raised against a fusion protein produced in Escherichia coli allowed the identification of a 122-kDa protein in X. campestris pv. vesicatoria cells expressing the avrBs3 gene. The antibody is specific for AvrBs3 in X. campestris pv. vesicatoria cells but also recognizes homologous proteins in other pathovars of X. campestris. We found that AvrBs3 is localized intracellularly in X. campestris pv. vesicatoria and is mainly in the soluble fraction. The effect of mutations in the hrp gene cluster on the function of AvrBs3 was examined. Expression of AvrBs3 in X. campestris pv. vesicatoria grown in minimal or complex medium is independent of the hrp gene cluster that determines pathogenicity and hypersensitivity to X. campestris pv. vesicatoria. In the plant, however, the hrp genes are required for elicitation of a race-specific resistance response.
Cloning, characterization, and DNA sequence of the rfaLK region for lipopolysaccharide synthesis in Salmonella typhimurium LT2
Authors: MacLachlan P R , Kadam S K , Sanderson K E
Published: November 2016

We have cloned and sequenced the rfaL and rfaK genes for lipopolysaccharide synthesis in Salmonella typhimurium LT2 on a 4.28-kb HindIII fragment from the previously described R' factor pKZ3 (S. K. Kadam, A. Rehemtulla, and K. E. Sanderson, J. Bacteriol. 161:277-284, 1985). rfaL is thought to encode a component of the O-antigen ligase, and rfaK is believed to encode the N-acetylglucosamine transferase. The genes were identified by the loss of complementation of prototype rfaL and rfaK mutations after Tn1000 mutagenesis. Translation of the nucleotide sequence predicted sizes of 45.9 and 43.1 kDa for the rfaL and rfaK gene products, respectively. Hydropathy analysis of the rfaL product suggested that it was an integral membrane protein. A third gene, rfaZ, was found to be an 808-bp open reading frame on the pyrE side of rfaK. Insertions into rfaZ reduced rfaK complementation, suggesting cotranscription in the pyrE-cysE direction. The rfaL gene is transcribed in the opposite direction in a separate operon which may also include rfaC. An incomplete open reading frame with homology to an Escherichia coli gene in the same region, rfaY, was found on the pyrE side of rfaZ. Complementation studies with Tn1000 insertions in rfaL showed that rfaL446 and rfaL447 are allelic. With the cloning of the rfaL and -K genes, the order of genes within the rfa cluster at 79 units on the linkage map was found to be cysE-rfaDFCLKZYJIBG-pyrE.
Cloning, sequencing, and expression of the Pseudomonas testosteroni gene encoding 3-oxosteroid delta 1-dehydrogenase
Authors: Plesiat P , Grandguillot M , Harayama S , Vragar S , Michel-Briand Y
Published: November 2016

Pseudomonas testosteroni ATCC 17410 is able to grow on testosterone. This strain was mutagenized by Tn5, and 41 mutants defective in the utilization of testosterone were isolated. One of them, called mutant 06, expressed 3-oxosteroid delta 1- and 3-oxosteroid delta 4-5 alpha-dehydrogenases only at low levels. The DNA region around the Tn5 insertion in mutant 06 was cloned into pUC19, and the 1-kbp EcoRI-BamHI segment neighbor to the Tn5 insertion was used to probe DNA from the wild-type strain. The probe hybridized to a 7.8-kbp SalI fragment. Plasmid pTES5, which is a pUC19 derivative containing this 7.8-kbp SalI fragment, was isolated after the screening by the 1-kbp EcoRI-BamHI probe. This plasmid expressed delta 1-dehydrogenase in Escherichia coli cells. The 2.2-kbp KpnI-KpnI segment of pTES5 was subcloned into pUC18, and pTEK21 was constructed. In E. coli containing the lacIq plasmid pRG1 and pTEK21, the expression of delta 1-dehydrogenase was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The induced level was about 40 times higher than the induced level in P. testosteroni. Delta 1-Dehydrogenase synthesized in E. coli was localized in the inner membrane fraction. The minicell experiments showed that a 59-kDa polypeptide was synthesized from pTEK21, and this polypeptide was located in the inner membrane fraction. The complete nucleotide sequence of the 2.2-kbp KpnI-KpnI segment of pTEK21 was determined. An open reading frame which encodes a 62.4-kDa polypeptide and which is preceded by a Shine-Dalgarno-like sequence was identified. The first 44 amino acids of the putative product exhibited significant sequence similarity to the N-terminal sequences of lipoamide dehydrogenases.
The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis
Authors: Ramakrishnan G , Zhao J L , Newton A
Published: November 2016

We have characterized flbF, a key locus located at the top of the flagellar gene hierarchy of Caulobacter crescentus. This gene is required for transcription from sigma 54 promoters of fla genes expressed late in the cell cycle. We have determined the nucleotide sequence of the gene, mapped the 5' end of the flbF RNA, and examined the pattern of expression in the cell cycle. Our results show that flbF is expressed earlier in the cell cycle than other fla genes, that it is expressed at a low level throughout the stalked cell cycle, and that its 5' regulatory region contains sequences that can be aligned with the sigma 28 promoter consensus reported for enteric bacteria. flbF contains an open reading frame of 700 residues with an amino-terminal half rich in hydrophobic residues that could correspond to six to eight transmembrane domains. The translated flbF sequence is very similar to LcrD (low calcium response) encoded by virulence plasmids of pathogenic Yersinia spp. (G. Plano, S. Barve, and S. Straley, J. Bacteriol. 173:7293-7303, 1991). LcrD and FlbF can be aligned over the entire length of the proteins with the greatest degree of sequence identity (45%) in the hydrophobic amino-terminal region. The high degree of sequence homology of proteins derived from widely differing organisms, including Caulobacter and Yersinia species, suggests that FlbF and LcrD may be representatives of a larger family of regulatory proteins with a common sensor mechanism for modifying responses to appropriate stimuli.
Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha
Authors: Mittenhuber G , Sonomoto K , Egert M , Friedrich C G
Published: November 2016

For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.
One-step cloning system for isolation of bacterial lexA-like genes
Authors: Calero S , Garriga X , Barbé J
Published: November 2016

A system to isolate lexA-like genes of bacteria directly was developed. It is based upon the fact that the presence of a lexA(Def) mutation is lethal to SulA+ cells of Escherichia coli. This system is composed of a SulA- LexA(Def) HsdR- strain and a lexA-conditional killer vector (plasmid pUA165) carrying the wild-type sulA gene of E. coli and a polylinker in which foreign DNA may be inserted. By using this method, the lexA-like genes of Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa, and P. putida were cloned. We also found that the LexA repressor of S. typhimurium presented the highest affinity for the SOS boxes of E. coli in vivo, whereas the LexA protein of P. aeruginosa had the lowest. Likewise, all of these LexA repressors were cleaved by the activated RecA protein of E. coli after DNA damage. Furthermore, under high-stringency conditions, the lexA gene of E. coli hybridized with the lexA genes of S. typhimurium and E. carotovora but not with those of P. aeruginosa and P. putida.
Differential response of the bvg virulence regulon of Bordetella pertussis to MgSO4 modulation
Authors: Scarlato V , Rappuoli R
Published: November 2016

Magnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon.
The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn-glycerophosphoryl diester phosphodiesterase
Authors: Ryffel C , Bucher R , Kayser F H , Berger-Bächi B
Published: November 2016

The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', and insertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphism among different staphylococcal isolates. We sequenced and characterized the hypervariable region of the methicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bp hypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNA sequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. The dru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open reading frame that codes for 145 amino acids (ORF145), whose deduced amino acid sequence showed 57% amino acid sequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) of Escherichia coli.
Molecular cloning of the extracellular endodextranase of Streptococcus salivarius
Authors: Lawman P , Bleiweis A S
Published: November 2016

We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.
Dosage of the smallest chromosome affects both the yeast-hyphal transition and the white-opaque transition of Candida albicans WO-1
Authors: McEachern M J , Hicks J B
Published: November 2016

The WO-1 strain of Candida albicans is capable of alternating between two highly distinct yeast cell types termed white and opaque (E. H. A. Rikkerrink, B. B. Magee, and P. T. Magee, J. Bacteriol. 170:895-899, 1988; B. Slutsky, M. Staebell, J. Anderson, L. Risen, M. Pfaller, and D. R. Soll, J. Bacteriol. 169:189-197, 1987). We have isolated WO-1 mutants that show a marked deficiency at being able to switch from the white form to the opaque form under conditions normally favorable for this transition. Pulsed-field electrophoresis demonstrated that one of the initial two spontaneous nonswitching mutants lacked the smallest chromosome that is normally present in WO-1. The availability of a WO-1 derivative whose only functional ADE2 gene is located on this small chromosome made possible, through the induction of chromosome nondisjunction, the isolation of numerous new mutants missing this chromosome as well as mutants containing two copies of the chromosome. Mutants missing the smallest chromosome showed a greatly diminished ability to produce opaque sectors and to produce germ tubes in the presence of human serum. Mutants containing two copies of the small chromosome showed an increased ability to produce germ tubes. These results indicate that this small chromosome carries one or more genes involved in both the white-opaque switch and the yeast-hyphal switch.
Temporal sequence of the recovery of traits during phenotypic curing of a Cytophaga johnsonae motility mutant
Authors: Gorski L , Leadbetter E R , Godchaux W
Published: November 2016

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.
Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii
Authors: Wolfinger E D , Bishop P E
Published: November 2016

The nucleotide sequence (3,600 bp) of a second copy of nifENX-like genes in Azotobacter vinelandii has been determined. These genes are located immediately downstream from vnfA and have been designated vnfENX. The vnfENX genes appear to be organized as a single transcriptional unit that is preceded by a potential RpoN-dependent promoter. While the nifEN genes are thought to be evolutionarily related to nifDK, the vnfEN genes appear to be more closely related to nifEN than to either nifDK, vnfDK, or anfDK. Mutant strains (CA47 and CA48) carrying insertions in vnfE and vnfN, respectively, are able to grow diazotrophically in molybdenum (Mo)-deficient medium containing vanadium (V) (Vnf+) and in medium lacking both Mo and V (Anf+). However, a double mutant (strain DJ42.48) which contains a nifEN deletion and an insertion in vnfE is unable to grow diazotrophically in Mo-sufficient medium or in Mo-deficient medium with or without V. This suggests that NifE and NifN substitute for VnfE and VnfN when the vnfEN genes are mutationally inactivated. AnfA is not required for the expression of a vnfN-lacZ transcriptional fusion, even though this fusion is expressed under Mo- and V-deficient diazotrophic growth conditions.
Genome conservation in isolates of Leptospira interrogans
Authors: Herrmann J L , Baril C , Bellenger E , Perolat P , Baranton G , Saint Girons I
Published: November 2016

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.
Induction of the alkA gene of Escherichia coli in gram-negative bacteria
Authors: Fernandez de Henestrosa A R , Barbé J
Published: November 2016

A broad-host-range plasmid containing a fusion of the alkA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria--33 species belonging to 19 genera--to study the induction of expression of the alkA gene by alkylating agents. The bacteria included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae, and Azotobacteraceae. Results obtained show that all bacteria tested, except Aeromonas hydrophila, Agrobacterium tumefaciens, Hafnia alvei, Rhizobium meliloti, Salmonella enteritidis, Xanthomonas campestris, and those of the genus Rhodobacter, are able to induce the alkA gene of E. coli in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. All these data indicate that the adaptive response to alkylating agents is present in bacterial species of several families and that the Ada box sequence must be widely conserved.
Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell
Authors: Stevens M K , Krause D C
Published: November 2016

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.
Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes
Authors: Metts J , West J , Doares S H , Matthysse A G
Published: November 2016

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.
Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli
Authors: Nara T , Lee L , Imae Y
Published: November 2016

The thermosensing ability of the Trg and Tap chemoreceptors in Escherichia coli was investigated after amplifying these receptors in a host strain lacking all four known chemoreceptors (Tar, Tsr, Trg, and Tap). Cells with an increased amount of either Trg or Tap showed mostly smooth swimming and no response to thermal stimuli. However, when the smooth-swimming bias of the cells was reduced by adding Trg- or Tap-mediated repellents, the cells showed clear changes in the swimming pattern upon temperature changes; Trg-containing cells showed tumbling at 23 degrees C but mostly smooth swimming at 32 degrees C, while Tap-containing cells showed smooth swimming at 20 degrees C but tumbling at 32 degrees C. These results indicate that although both Trg and Tap have the ability to sense thermal stimuli, Trg functions as a warm receptor, as reported previously for Tar and Tsr, while Tap functions as a cold receptor.
A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion
Authors: Bell-Pedersen D , Galloway Salvo J L , Belfort M
Published: November 2016

A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene. Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo. Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts. Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts. This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E. coli were attributable to the presence of the overlapping transcript. Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement. This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.
Mutagenesis and isolation of Aeromonas hydrophila genes which are required for extracellular secretion
Authors: Bo J N , Howard S P
Published: November 2016

Transposon mutagenesis was used to isolate mutants of Aeromonas hydrophila which were deficient in the production of extracellular proteins. The culture supernatants of two of the mutants were essentially devoid of the proteins normally secreted by the parent strain, despite their continued synthesis. Western immunoblot analysis of one of these proteins indicated that normal signal sequence processing occurred but that normal zymogen activation did not, and cell fractionation experiments indicated that both mutants accumulated the three different extracellular proteins assayed in a position external to the cytoplasmic membrane, presumably in the periplasm. The two mutants differed, however, in that one was lysed during the osmotic shock procedures and also contained severely reduced amounts of two of the major protein components of the outer membrane. The wild-type chromosomal regions into which the transposon had been inserted in the two mutants were cloned. In each case, transconjugants of the mutants containing the corresponding cloned fragment were complemented for the defects in secretion, and one of the mutants was complemented by the heterologous clone as well, suggesting the possibility of an interaction between these two genes or gene products. These results indicate that two separate functions which are required for extracellular secretion were interrupted in the insertion mutants and that one of these is also critically important in the biogenesis of the outer membrane.
Novel organization of the common nodulation genes in Rhizobium leguminosarum bv. phaseoli strains
Authors: Vázquez M , Dávalos A , de las Peñas A , Sánchez F , Quinto C
Published: November 2016

Nodulation by Rhizobium, Bradyrhizobium, and Azorhizobium species in the roots of legumes and nonlegumes requires the proper expression of plant genes and of both common and specific bacterial nodulation genes. The common nodABC genes form an operon or are physically mapped together in all species studied thus far. Rhizobium leguminosarum bv. phaseoli strains are classified in two groups. The type I group has reiterated nifHDK genes and a narrow host range of nodulation. The type II group has a single copy of the nifHDK genes and a wide host range of nodulation. We have found by genetic and nucleotide sequence analysis that in type I strain CE-3, the functional common nodA gene is separated from the nodBC genes by 20 kb and thus is transcriptionally separated from the latter genes. This novel organization could be the result of a complex rearrangement, as we found zones of identity between the two separated nodA and nodBC regions. Moreover, this novel organization of the common nodABC genes seems to be a general characteristic of R. leguminosarum bv. phaseoli type I strains. Despite the separation, the coordination of the expression of these genes seems not to be altered.
N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides
Authors: Parales J , Greenberg E P
Published: November 2016

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.
Intracellular location of the autolytic N-acetylmuramyl-L-alanine amidase in Bacillus subtilis 168 and in an autolysis-deficient mutant by immunoelectron microscopy
Authors: Hobot J A , Rogers H J
Published: November 2016

Antisera against purified autolytic N-acetylmuramyl-L-alanine amidase from Bacillus subtilis 168 were prepared in rabbits. They neutralized the enzymatic action of the purified amidase acting on isolated sodium dodecyl sulfate (SDS)-treated walls from the same organism. They also inhibited the lysis of native walls, but only after the walls lysed partially. Amidase adsorbed to insoluble walls still combined with antibody. Antisera did not stop the lysis of whole cells. Lowicryl HM20 sections of both strain 168 and its autolytic mutant strain FJ6 were prepared by the progressive-lowering-of-temperature technique, immunolabeled with the antisera, and visualized with colloidal gold particles as markers. The highest concentration of gold particles seemed to be in the septa of dividing cells, followed by the side walls. There was some labeling of the cytoplasm. Adsorption of sera with SDS-treated walls reduced the overall labeling of sections considerably but did not alter the relative intracellular distribution of particles. The results for strains 168 and FJ6 were similar. Labeling of SDS-treated walls unexpectedly revealed the presence of a wall-bound amidase fraction.
Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription
Authors: Wozniak D J , Ohman D E
Published: November 2016

Most strains of Pseudomonas aeruginosa isolated from the respiratory tracts of cystic fibrosis patients have a mucoid colony morphology due to the synthesis of an expolysaccharide called alginate. The algB gene product (AlgB) is necessary for the high-level production of alginate in mucoid P. aeruginosa. In this study, AlgB was shown to be involved in the transcription of algD, a gene previously demonstrated to be activated in mucoid P. aeruginosa. In vitro and in vivo expression studies reveal that algB encodes a protein with a molecular size of 49 kDa. The DNA sequence of a 2.2-kb P. aeruginosa fragment containing algB was also determined. The amino-terminal domain of AlgB was found to be conserved with the amino-terminal domains of the response regulator class of two-component regulatory proteins. The central domain of AlgB has sequences highly conserved with those in the NtrC subfamily of transcriptional activators (NtrC, NifA, HydG, DctD, FlbD, TyrR, and PgtA). The central domain of AlgB also contains a potential nucleotide binding site. AlgB is the first NtrC homolog described from P. aeruginosa. At the carboxy terminus of AlgB, a helix-turn-helix motif was observed, suggesting that AlgB is a DNA-binding protein. The strongly conserved NtrC-like central domain of AlgB is not present in AlgR, another alginate response regulator. This study therefore identifies and characterizes the second of at least two unique response regulators used by P. aeruginosa to control alginate gene expression.
Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg
Authors: Wohlfarth G , Geerligs G , Diekert G
Published: November 2016

The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.
Salmonella typhimurium histidine periplasmic permease mutations that allow transport in the absence of histidine-binding proteins
Authors: Speiser D M , Ames G F
Published: November 2016

Periplasmic transport systems consist of a membrane-bound complex and a periplasmic substrate-binding protein and are postulated to function by translocating the substrate either through a nonspecific pore or through specific binding sites located in the membrane complex. We have isolated mutants carrying mutations in one of the membrane-bound components of the histidine permease of Salmonella typhimurium that allow transport in the absence of both histidine-binding proteins HisJ and LAO (lysine-, arginine-, ornithine-binding protein). All of the mutations are located in a limited region of the nucleotide-binding component of the histidine permease, HisP. The mutants transported substrate in the absence of binding proteins only when the membrane-bound complex was produced in large amounts. At low (chromosomal) levels, the mutant complex was unable to transport substrate in the absence of binding proteins but transported it efficiently in the presence of HisJ. The alterations responsible for the mutations were identified by DNA sequencing; they are closely related to a group of hisP mutations isolated as suppressors of HisJ interaction mutations (G. F.-L. Ames and E. N. Spudich, Proc. Natl. Acad. Sci. USA 73:1877-1881, 1976). The hisP suppressor mutations behaved similarly to these newly isolated mutations despite the entirely different selection procedure. The results are consistent with the HisP protein carrying or contributing to the existence of a substrate-binding site that can be mutated to function in the absence of a binding protein.
Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its gene product
Authors: Gassmann G S , Jacobs E , Deutzmann R , Göbel U B
Published: November 2016

The fla gene of Borrelia burgdorferi GeHo was analyzed and expressed in Escherichia coli. The structural gene encodes a flagellar protein of 336 amino acids. Comparative sequence analysis of the amino acid sequence revealed a high degree of sequence conservation with flagellins from both phylogenetically related and unrelated bacteria. The antigenic properties of the B. burgdorferi Fla protein were studied by synthesizing overlapping octapeptides, which were screened by using a battery of different monoclonal and polyclonal antibodies from various species directed against native and denatured flagellar proteins. No single species-independent immunodominant epitope could be located. However, immunoreactive oligopeptides clustered within the variable middle region (N-180 to I-260). This region could constitute a candidate antigen for more specific and sensitive serodiagnosis of Lyme borreliosis.
Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii
Authors: Cai J , Speed R R , Winkler H H
Published: November 2016

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.
Thioredoxin elicits a new dihydrolipoamide dehydrogenase activity by interaction with the electron-transferring flavoprotein in Clostridium litoralis and Eubacterium acidaminophilum
Authors: Meyer M , Dietrichs D , Schmidt B , Andreesen J R
Published: November 2016

The glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing dihydrolipoamide. The first one was found to be a genuine dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by dihydrolipoamide was the strongly preferred reaction. A second dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088-2095, 1990) and a thioredoxin. This enzyme system was responsible for most of the dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.
Transfer of Tn925 and plasmids between Bacillus subtilis and alkaliphilic Bacillus firmus OF4 during Tn925-mediated conjugation
Authors: Guffanti A A , Quirk P G , Krulwich T A
Published: November 2016

Conjugative transposon Tn925 was transferred to alkaliphilic Bacillus firmus OF4 during mating experiments, as monitored by the acquisition of tetracycline resistance at pH 7.5 and confirmed by Southern analysis of chromosomal DNA from transconjugants. Tetracycline resistance could not be demonstrated at pH 10.5, but transconjugants retained resistance upon growth at pH 7.5 after having grown for several generations at pH 10. When the Bacillus subtilis donor strain contained plasmids, either pUB110 or pTV1, in addition to Tn925, transfer of the plasmid to the alkaliphile occurred during conjugation, either together with or independently of the transfer of the transposon. The plasmids were stable in B. firmus OF4, expressing their resistance markers for kanamycin or chloramphenicol at pH 7.5 after growth of the transformants at high pH. Transconjugant B. firmus OF4, which carried Tn925, could serve as the donor in mating experiments with B. subtilis lacking the transposon. These studies establish a basis for initiation of genetic studies in this alkaliphilic Bacillus species, including the introduction of cloned genes and the use of transposon-mediated insertional mutagenesis.
RcsA, an unstable positive regulator of capsular polysaccharide synthesis
Authors: Stout V , Torres-Cabassa A , Maurizi M R , Gutnick D , Gottesman S
Published: November 2016

RcsA is an unstable positive regulator required for the synthesis of colanic acid capsular polysaccharide in Escherichia coli. Degradation of the RcsA protein in vivo depends on the ATP-dependent Lon protease. DNA sequence analysis of the rcsA gene reveals a single open reading frame for a 23,500-Da highly basic protein (pI = 9.9), consistent with the observed size of the purified subunit of RcsA. The DNA and protein sequences are highly homologous to the rcsA gene and protein from Klebsiella pneumoniae and other species. The carboxy-terminal region of RcsA contains a possible helix-turn-helix DNA-binding motif that resembles sequences found at the carboxy terminus of RcsB, another positive regulator of capsule synthesis, and in several other transcriptional regulators including members of the LuxR family. rcsA62, a mutation in rcsA that leads to increased capsule synthesis, encodes a protein designated RcsA*, which differs from wild-type RcsA only in the replacement of Met-145 by valine. The RcsA* protein is subject to Lon-dependent degradation. The stability of wild-type RcsA in vivo is increased by multicopy RcsB. Conversely, RcsA is degraded more rapidly in rcsB mutant hosts than in wild-type hosts. These results suggest that RcsA and RcsB interact in vivo and are consistent with genetic experiments that indicate an interaction between RcsA and RcsB. Based on these experiments, we propose a model for capsule regulation in which RcsA interacts directly with RcsB to promote transcription of the genes for capsule synthesis.