bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene
Authors: Narberhaus F , Giebeler K , Bahl H
Published: November 2016

The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors. By using the E. coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C. acetobutylicum, respectively, were isolated. The cloned fragments partially overlapped, and together they spanned 4,083 bp of the clostridial genome that were completely sequenced. On one strand, four open reading frames of which the last was obviously truncated were identified. The last three genes showed high homology to the grpE, dnaK, and dnaJ heat shock genes of E. coli, respectively. They were preceded by an open reading frame (orfA) without any homology to sequences available in the EMBL or GenBank data bases. Typical translational start sites could be found in front of all four genes. Northern (RNA) blot analysis revealed transcripts of this region with a maximum length of 5.0 kb. Thus, these genes are probably organized in an operon. A transcription terminator could be found between the dnaK and dnaJ genes. By primer extension analysis, a major heat-inducible transcription start site was identified 49 bases upstream of orfA. This site was preceded by a region (5'-TTGACA[17 bp]TATTTT) that exhibited high homology to the consensus promoter sequences of gram-positive bacteria as well as sigma 70-dependent E. coli. Between this promoter and the initiation codon of orfA, a hairpin-loop structure with a possible regulatory role in the expression of these genes was found. Additional heat-inducible transcription start sites were located 69 bases upstream of orfA and 87 bases upstream of grpE; the corresponding promoter regions showed less similarity to other known promoter sequences. Maximum mRNA levels of this heat shock operon were found about 15 min after a heat shock from 30 to 42 degrees C. Our results indicate that orfA codes for an unknown heat shock protein.
Identification of heat-stable A-factor from Myxococcus xanthus
Authors: Kuspa A , Plamann L , Kaiser D
Published: November 2016

The asg mutants of Myxococcus xanthus fail to produce a set of related substances called A-factor. A-factor is released into the medium and is required early in fruiting body development. Lacking A-factor, the asg mutants are defective in aggregation, sporulation, and expression of most genes whose products appear later than 1 h after development is induced by starvation. Previous work has shown that these defects are reversed when A-factor, released by developing wild-type cells, is added to asg mutant cells. Part of the material in conditioned medium with A-factor activity is heat stable and dialyzable. This low-molecular-weight A-factor consists of a mixture of amino acids and peptides. Fifteen single amino acids have A-factor activity, and 11 of these are found in conditioned medium. Mixtures of amino acids have a total activity approximately equal to the sum of the activities of their constituents. Conditioned medium also contains peptides with A-factor activity. Pure peptides have A-factor activity, and their specific activities are equal to or less than the sum of the activities of their constituent amino acids. There is no evidence for a specialized A-factor peptide in conditioned medium, one with a specific activity greater than the sum of its constituent amino acids. About half of the heat-stable A-factor activity in conditioned medium can be accounted for by free amino acids, and the remaining half can be accounted for by peptides. It is argued that heat-stable A-factor induces A-dependent gene expression not by the nutritional action of amino acids but through a chemosensory circuit.
Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species
Authors: Helander I M , Hurme R , Haikara A , Moran A P
Published: November 2016

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H2O; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the O antigen. The major fatty acids were ester- and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H2O and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different O antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic O-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic O-6,7 antigen, was exclusively LPS-H2O. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.
Effects of inducing expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase
Authors: Monticello R A , Angov E , Brusilow W S
Published: November 2016

To evaluate whether expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase is sufficient to cause membrane proton permeability, plasmids carrying different combinations of the uncB, E, and F genes, encoding the a, c, and b subunits of the F0 sector, cloned behind the inducible lac promoter in pUC9 or pUC18, were constructed. The effects of inducing F0 synthesis in an unc deletion strain were monitored by measuring cell growth rate, quantitating F0 subunits by immunoblotting, and measuring the ability of membranes to maintain a respiration-induced proton gradient and to bind F1 and carry out energy-coupling reactions. The levels of functional reconstitutable F0 in membranes could be increased four- to sixfold with no change in cellular growth rate or membrane proton permeability (assayed by fluorescence quenching). These results were obtained in uninduced cultures, so the F0 genes were presumably being transcribed from some promoter besides lac. Induction of transcription of all three F0 genes produced increased amounts of F0 subunits in membranes as determined by immunoblot and F1-binding assays, but, when reconstituted with F1, the F0 in membranes isolated from induced cultures was significantly less functional than the F0 in membranes isolated from uninduced cultures. Such induction did result in growth inhibition, but there was no correlation between growth inhibition and either increased membrane proton permeability or the presence of functional, reconstitutable F0.
Isolation of DNA damage-inducible promoters in Escherichia coli: regulation of polB (dinA), dinG, and dinH by LexA repressor
Authors: Lewis L K , Jenkins M E , Mount D W
Published: November 2016

A new genetic screening method has been developed to isolate Escherichia coli promoters which are components of the SOS regulon. Plasmids containing the regulatory regions of polB (dinA) and two new loci, dinG and dinH, were characterized. Galactokinase gene fusion experiments indicated that transcription of these genes is inducible by treatment with mitomycin and conforms to a classical model of SOS regulation involving simple LexA repression. Mapping studies using the E. coli DNA library of Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) revealed that dinG and dinH are located at 17.8 and 19.8 min on the chromosome, respectively. The nucleotide sequence of the dinH regulatory region contains a segment which is very similar to previously characterized binding sites for LexA protein. An asymmetric, noncanonical 20-bp LexA operator in the cloned dinG promoter region was identified. Additional experiments have revealed that the nucleotide sequence of the gene immediately downstream of the DNA damage-inducible polB locus encodes a polypeptide which has extensive sequence homology to several known and putative DNA and RNA helicase proteins. This gene, which is not regulated by the LexA repressor, has been designated hepA. The predicted amino acid sequence of the product of hepA contains several highly conserved sequence motifs that are also found in enzymes such as the RecQ and UvrB proteins of E. coli and the Rad3 protein of Saccharomyces cerevisiae.
Physiological functions of hydroperoxidases in Rhodobacter capsulatus
Authors: Hochman A , Figueredo A , Wall J D
Published: November 2016

Rhodobacter capsulatus J1 has two hydroperoxidases: a catalase-peroxidase and a peroxidase. A mutant strain, AH18, that had no catalase-peroxidase was isolated. The growth rate under aerobic and photosynthetic conditions, respiration, superoxide dismutase and peroxidase activities, and pigment content of the mutant were similar to those of the wild type. AH18 was more susceptible to killing and to inhibition of nitrogenase by H2O2 but not by molecular oxygen. The incidences of spontaneous mutations were similar in both strains. Viable counts in aerobic but not anaerobic cultures of AH18 started to decline as soon as the cultures reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. It is inferred that the peroxidase provides protection against H2O2 in log-phase cells and that the catalase-peroxidase provides protection under the oxidative conditions that prevail in aging cultures. This protective function might be related to the dual activity of the latter as a catalase and a peroxidase or to its capacity to oxidize NADH, NADPH, and cytochrome c.
Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants
Authors: Urzainqui A , Walker G C
Published: November 2016

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.
Chromosome-mediated 2,3-dihydroxybenzoic acid is a precursor in the biosynthesis of the plasmid-mediated siderophore anguibactin in Vibrio anguillarum
Authors: Chen Q , Actis L A , Tolmasky M E , Crosa J H
Published: November 2016

We have isolated a recombinant clone harboring the chromosomal aroC gene, encoding chorismate synthase, from Vibrio anguillarum 775 by complementation of the Escherichia coli aroC mutant AB2849 which was transfected with a cosmid gene bank of the plasmidless V. anguillarum H775-3. The nucleotide sequence was determined, and an open reading frame that corresponds to a protein of 372 amino acids was found. The calculated mass of 40,417 Da was correlated with the size of the V. anguillarum aroC product detected in vitro. The homology of the V. anguillarum aroC gene to the aroC genes of E. coli and Salmonella typhi is 68% at the nucleotide level and 78% at the protein level. The expression of the aroC transcript is not regulated by iron, as determined by Northern (RNA) blot hybridization analysis. After insertion of an antibiotic resistance gene cassette within the cloned aroC gene, an aroC mutant of V. anguillarum was generated by allelic exchange. This mutant is deficient in the production of 2,3-dihydroxybenzoic acid (2,3-DHBA). Our bioassay and complementation experiments with this mutant demonstrate that the chromosome-mediated 2,3-DHBA is a precursor of the pJM1 plasmid-mediated siderophore anguibactin.
Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4
Authors: Balzer D , Pansegrau W , Lanka E
Published: November 2016

Two essential transfer genes of the conjugative plasmid RP4 were altered by site-directed mutagenesis: traG of the primase operon and traI of the relaxase operon. To evaluate effects on the transfer phenotype of the point mutations, we have reconstituted the RP4 transfer system by fusion of the transfer regions Tra1 and Tra2 to the small multicopy replicon ColD. Deletions in traG or traI served to determine the Tra phenotype of mutant plasmids by trans complementation. Two motifs of TraG which are highly conserved among TraG-like proteins in several other conjugative DNA transfer systems were found to be essential for TraG function. One of the motifs resembles that of a nucleotide binding fold of type B. The relaxase (TraI) catalyzes the specific cleaving-joining reaction at the transfer origin needed to initiate and terminate conjugative DNA transfer (W. Pansegrau, W. Schröder, and E. Lanka, Proc. Natl. Acad. Sci. USA 90:2925-2929, 1993). Phenotypes of mutations in three motifs that belong to the active center of the relaxase confirmed previously obtained biochemical evidence for the contributions of the motifs to the catalytic activity of TraI. Expression of the relaxase operon is greatly increased in the absence of an intact TraI protein. This finding suggests that the relaxosome which assembles only in the presence of the TraI in addition to its enzymatic activity plays a role in gene regulation.
Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors
Authors: Peters E A , Schatz P J , Johnson S S , Dower W J
Published: November 2016

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.
Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli
Authors: Ohyama T , Igarashi K , Kobayashi H
Published: November 2016

Ohyama et al. previously isolated Escherichia coli mutant RS1, which had a negligible activity for sodium ion extrusion at alkaline pH (T. Ohyama, R. Imaizumi, K. Igarashi, and H. Kobayashi, J. Bacteriol. 174:7743-7749, 1992). Our present study showed that the mutation of RS1 was compensated for by a cloned chaA gene. It has been proposed that sodium ion extrusion by ChaA is prevented under physiological conditions (D. M. Ivey, A. A. Guffanti, J. Zemsky, E. Pinner, R. Karpel, E. Padan, S. Schuldiner, and T. A. Krulwich, J. Biol. Chem. 268:11296-11303, 1993). In order to clarify the physiological role of chaA in sodium ion circulation at alkaline pH, we constructed a delta chaA mutant. The resultant mutant, TO112, deficient in both nhaA and chaA, was unable to grow at pH 8.5 in medium containing 0.1 M sodium chloride and had negligible sodium ion extrusion activity. However, TO112 grew at pH 7.0 in medium containing 0.4 M sodium chloride. Sodium ions were extruded from TO112 cells at neutral pH. The extrusion activity at pH 7.5 was greatly reduced by the deletion of nhaB. These data demonstrate that the activity of nhaB is low at high pH and that ChaA extrudes sodium ions at alkaline pH. The uptake of calcium ions by everted membrane vesicles prepared from the delta chaA mutant TO110 was 60% of the activity observed in the vesicles of the wild-type strain at pH 8.5, but the activity at neutral pH was not reduced by the deletion of chaA. Therefore, it was also suggested that ChaA plays a role in calcium ion circulation at alkaline pH.
Isolation and biochemical characterization of a novel lantibiotic mutacin from Streptococcus mutans
Authors: Novák J , Caufield P W , Miller E J
Published: November 2016

Certain members of the indigenous biota of humans produce antimicrobial substances called bacteriocins, which inhibit other bacteria, including members of their own species. One of these substances, mutacin, is made by Streptococcus mutans, a member of the oral biota. Mutacin inhibits other mutans streptococci as well as many gram-positive exogenous pathogens. Here, we report for the first time the purification and partial biochemical characterization of a lanthionine-containing mutacin peptide from S. mutants T8. The biologically active peptide was isolated from the broth cultures by ultrafiltration and differential precipitation. The final mutacin preparation was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. A molecular mass of the peptide was estimated by electrospray ionization mass spectroscopy to be 3,244.64 +/- 1.15 Da. Its amino acid composition indicates the presence of lanthionine and likely beta-methyllanthionine in a total of about 25 amino acids. Because alpha,beta-unsaturated amino acids, the precursors of lanthionine residues, are often found in lantibiotics, we carried out the addition reaction of the mutacin with N-(methyl)mercaptoacetamide. The subsequent electrospray ionization mass spectroscopy analysis indicated the presence of two reaction products with M(r)s of 3,350.45 and 3,456.0. These are interpreted as the mutacin molecule with the addition of one and two molecules of reagent to the unsaturated amino acids, respectively. Sequencing of the peptide revealed an N-terminal amino acid sequence of Asn-Arg-Trp-Trp-Gln-Gly-Val-Val.
Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli
Authors: Mengin-Lecreulx D , Blanot D , van Heijenoort J
Published: November 2016

In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine. The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used. Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid. Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi. For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed. The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values. Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis. The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides. HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi.
Molecular analysis of the rfb gene cluster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains
Authors: Xiang S H , Hobbs M , Reeves P R
Published: November 2016

The Salmonella enterica O antigen is a highly variable surface polysaccharide composed of a repeated oligosaccharide (the O unit). The O unit produced by serogroup D2 has structural features in common with those of groups D1 and E1, and hybridization studies had previously suggested that the D2 rfb gene cluster responsible for O-unit biosynthesis is indeed a hybrid of the two. In this study, the rfb gene cluster was cloned from a group D2 strain of S. enterica sv. Strasbourg. Mapping, hybridization, and DNA sequencing showed that the organization of the D2 rfb genes is similar to that of group D1, with the alpha-mannosyl transferase gene rfbU replaced by rfbO, the E1-specific beta-mannosyl transferase gene. The E1-specific polymerase gene (rfc) has also been acquired. Interestingly, the D1-like and E1-like rfb regions are separated by an additional sequence closely related to an element (Hinc repeat [H-rpt]) associated with the Rhs loci of Escherichia coli. The H-rpt resembles an insertion sequence and possibly mediated the intraspecific recombination events which produced the group D2 rfb gene organization.
Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2
Authors: Menon N K , Chatelus C Y , Dervartanian M , Wendt J C , Shanmugam K T , Peck H D , Przybyla A E
Published: November 2016

The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced. They occur in an operon (hyb) which contains seven open reading frames. An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1. All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3. The hyb operon was mapped at 65 min on the E. coli chromosome.
Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria
Authors: Cirillo J D , Weisbrod T R , Banerjee A , Bloom B R , Jacobs W R
Published: November 2016

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.
Requirements for mobilization of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB and RP4 traG are interchangeable
Authors: Cabezón E , Lanka E , de la Cruz F
Published: November 2016

Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB. traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388. This result suggests a dual specificity of TrwB-like proteins in conjugation. The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1.
Transformation of Mycoplasma gallisepticum with Tn916, Tn4001, and integrative plasmid vectors
Authors: Cao J , Kapke P A , Minion F C
Published: November 2016

Mycoplasma gallisepticum causes respiratory disease in avian species, but little is known about its mechanism(s) of pathogenesis. These studies were undertaken in order to develop genetic systems for analysis of potential virulence factors. M. gallisepticum was transformed with plasmids containing one of the gram-positive transposons Tn916 or Tn4001, which inserted randomly into the mycoplasmal chromosome. Plasmids containing cloned chromosomal DNA were also constructed and tested for integration into regions of DNA homology derived either from chromosomal fragments or from the gentamicin resistance marker from Tn4001. These studies demonstrate that M. gallisepticum is amenable to transformation with both transposons and integrative vectors.
Response to UV damage by four Escherichia coli K-12 restriction systems
Authors: Kelleher J E , Raleigh E A
Published: November 2016

To understand the role of restriction in regulating gene flow in bacterial populations, we would like to understand the regulation of restriction enzyme activity. Several antirestriction (restriction alleviation) systems are known that reduce the activity of type I restriction enzymes like EcoKI in vivo. Most of these do not act on type II or type III enzymes, but little information is available for the unclassified modification-dependent systems, of which there are three in E. coli K-12. Of particular interest are two physiological controls on type I enzymes: EcoKI restriction is reduced 2 to 3 orders of magnitude following DNA damage, and a similar effect is seen constitutively in Dam- cells. We used the behavior of EcoKI as a control for testing the response to UV treatment of the three endogenous modification-dependent restriction systems of K-12, McrA, McrBC, and Mrr. Two of these were also tested for response to Dam status. We find that all four resident restriction systems show reduced activity following UV treatment, but not in a unified fashion; each response was genetically and physiologically distinct. Possible mechanisms are discussed.
Transcriptional analysis of the Pseudomonas aeruginosa genes algR, algB, and algD reveals a hierarchy of alginate gene expression which is modulated by algT
Authors: Wozniak D J , Ohman D E
Published: November 2016

Strains of Pseudomonas aeruginosa which colonize and infect the lungs of cystic fibrosis patients have a mucoid colony morphology due to the overproduction of the exopolysaccharide alginate. The response regulators AlgB and AlgR are required for the transcription of algD, a tightly regulated gene encoding GDP-mannose dehydrogenase, which is critical for P. aeruginosa alginate biosynthesis. Previous studies indicated that mutations in the algT gene of mucoid FRD1 P. aeruginosa result in nonmucoid derivatives. However, the specific role for algT in alginate gene regulation has not been elucidated. In this study, transcription of algB, algD, and algR was characterized by gene fusion and primer extension analysis. Expression of algR and algD was abolished in P. aeruginosa strains containing algT::Tn501 insertions because of lack of transcription initiation at the algR and algD promoters. An algR mutation was constructed in FRD1, and this resulted in the loss of alginate production and a dramatic decrease in algD transcription. RNA and gene fusion analysis revealed that algB is not required for algR expression, nor is algR necessary for transcription of algB. Thus, with the exception of a requirement for AlgT, the AlgB and AlgR pathways appear to be independent of each other. In gel band mobility shift assays, a protein(s) present in extracts from mucoid and algB and algR mutant P. aeruginosa strains formed a specific complex with algD sequences located immediately upstream of the start of transcription. No binding to these sequences was observed when extracts from algT mutant strains were examined. A model proposed suggests that a hierarchy of alginate gene expression exists in which AlgT is required for transcription of the response regulators algB and algR, which in turn are necessary for algD expression. AlgT or a protein under algT control also binds to sequences located within the algD promoter.
Nucleoid partitioning and the division plane in Escherichia coli
Authors: Woldringh C L , Zaritsky A , Grover N B
Published: November 2016

Escherichia coli nucleoids were visualized after the DNA of OsO4-fixed but hydrated cells was stained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). In slowly growing cells, the nucleoids are rod shaped and seem to move along the major cell axis, whereas in rapidly growing, wider cells they consist of two- to four-lobed structures that often appear to advance along axes lying perpendicular or oblique to the major axis of the cell. To test the idea that the increase in cell diameter following nutritional shift-up is caused by the increased amount of DNA in the nucleoid, the cells were subjected to DNA synthesis inhibition. In the absence of DNA replication, the nucleoids continued to move in the growing filaments and were pulled apart into small domains along the length of the cell. When these cells were then transferred to a richer medium, their diameters increased, especially in the region enclosing the nucleoid. It thus appears that the nucleoid motive force does not depend on DNA synthesis and that cell diameter is determined not by the amount of DNA per chromosome but rather by the synthetic activity surrounding the nucleoid. Under the non-steady-state but balanced growth conditions induced by thymine limitation, nucleoids become separated into small lobules, often lying in asymmetric configurations along the cell periphery, and oblique and asymmetric division planes occur in more than half of the constricting cells. We suggest that such irregular DNA movement affects both the angle of the division plane and its position.
DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect
Authors: Williams S G , Egan J B
Published: November 2016

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.
Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis
Authors: Rey L , Imperial J , Palacios J M , Ruiz-Argüeso T
Published: November 2016

The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argüeso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 +/- 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency. Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. leguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. leguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.
Substrate specificity of catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida and its relationship to cell growth
Authors: Cerdan P , Wasserfallen A , Rekik M , Timmis K N , Harayama S
Published: November 2016

Catechol 2,3-dioxygenase encoded by TOL plasmid pWW0 of Pseudomonas putida consists of four identical subunits, each containing one ferrous ion. The enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. Two mutants of catechol 2,3-dioxygenases (4ECR1 and 4ECR6) able to oxidize 4-ethylcatechol, one mutant (3MCS) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and 4-methylcatechol, and one phenotypic revertant of 3MCS (3MCR) which had regained the ability to oxidize 3-methylcatechol were characterized by determining their Km and partition ratio (the ratio of productive catalysis to suicide catalysis). The amino acid substitutions in the four mutant enzymes were also identified by sequencing their structural genes. Wild-type catechol 2,3-dioxygenase was inactivated during the catalysis of 4-ethylcatechol and thus had a low partition ratio for this substrate, whereas the two mutant enzymes, 4ECR1 and 4ECR6, had higher partition ratios for it. Similarly, mutant enzyme 3MCS had a lower partition ratio for 3-methylcatechol than that of 3MCR. Molecular oxygen was required for the inactivation of the wild-type enzyme by 4-ethylcatechol and of 3MCS by 3-methylcatechol, and the inactivated enzymes could be reactivated by incubation with FeSO4 plus ascorbic acid. The enzyme inactivation is thus most likely mechanism based and occurred principally by oxidation and/or removal of the ferrous ion in the catalytic center. In general, partition ratios for catechols lower than 18,000 did not support bacterial growth. A possible meaning of the critical value of the partition ratio is discussed.
Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae
Authors: Tang L , Zhang Y X , Hutchinson C R
Published: November 2016

Targeted inactivation of the valine (branched-chain amino acid) dehydrogenase gene (vdh) was used to study the role of valine catabolism in the production of tylosin in Streptomyces fradiae and spiramycin in Streptomyces ambofaciens. The deduced products of the vdh genes, cloned and sequenced from S. fradiae C373.1 and S. ambofaciens ATCC 15154, are approximately 80% identical over all 363 amino acids and 96% identical over a span of the first N-terminal 107 amino acids, respectively, to the deduced product of the Streptomyces coelicolor vdh gene. The organization of the regions flanking the vdh genes is the same in all three species. Inactivation of the genomic copy of the vdh gene in S. fradiae and S. ambofaciens by insertion of a hygromycin resistance (hyg) gene caused loss of the valine dehydrogenase (Vdh) activity, and thus only one enzyme is responsible for the Vdh activity in these organisms. Analysis of the culture broth by bioassay revealed that the vdh::hyg mutants produce an approximately sixfold-lower level of tylosin and an approximately fourfold-lower level of spiramycin than the wild-type S. fradiae and S. ambofaciens strains, while maintaining essentially identical growth in a defined minimal medium with either 25 mM ammonium ion or 0.05% asparagine as the nitrogen source. The addition of the valine catabolite, propionate or isobutyrate, and introduction of the wild-type vdh gene back to each vdh::hyg mutant reversed the negative effect of the vdh::hyg mutation on spiramycin and tylosin production. These data show that the catabolism of valine is a major source of fatty acid precursors for macrolide biosynthesis under defined growth conditions and imply that amino acid catabolism is a vital source of certain antibiotic precursors in actinomycetes.
Transmembrane pH of Clostridium acetobutylicum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased
Authors: Girbal L , Vasconcelos I , Soucaille P
Published: November 2016

Evidence is reported here that alkalinization of Clostridium acetobutylicum cytoplasm involves hydrogenase activity. A decrease of in vivo hydrogenase activity is accompanied by intracellular accumulation of protons leading to a negative (interior acidic) pH gradient. However, the organism is able to maintain a constant proton motive force by interconverting chemical and electrical potentials.
Role of signal peptides in targeting of proteins in cyanobacteria
Authors: Mackle M M , Zilinskas B A
Published: November 2016

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.
Circadian rhythms of cyanobacteria: monitoring the biological clocks of individual colonies by bioluminescence
Authors: Kondo T , Ishiura M
Published: November 2016

Reproducible circadian rhythms of bioluminescence from individual colonies of cyanobacteria (Synechococcus sp. strain PCC 7942) has been observed. Phenotypic monitoring of colonies on agar plates will enable us to genetically analyze the molecular mechanism of the circadian clock of cyanobacteria by screening for clock mutants. By the introduction of a bacterial luciferase gene, we previously developed a transformed cyanobacterial strain (AMC149) that expresses luciferase as a bioluminescent reporter of the circadian clock. In liquid culture, AMC149 expresses a rhythm of bioluminescence that displays the same behavior as circadian rhythms in higher eukaryotes. Improvements in the technique for administering the reporter enzyme's substrate (decanal) and a highly sensitive photon-counting camera allow monitoring the bioluminescence of single colonies. Individual colonies on agar plates displayed a rhythmicity which is essentially the same as that previously reported for liquid cultures.
The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide
Authors: Akatsuka H , Kawai E , Omori K , Komatsubara S , Shibatani T , Tosa T
Published: November 2016

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.
Competition between Vibrio fischeri strains during initiation and maintenance of a light organ symbiosis
Authors: Lee K H , Ruby E G
Published: November 2016

Colonization of the light-emitting organ of the Hawaiian squid Euprymna scolopes is initiated when the nascent organ of a newly hatched squid becomes inoculated with Vibrio fischeri cells present in the ambient seawater. Although they are induced for luminescence in the light organ, these symbiotic strains are characteristically non-visibly luminous (NVL) when grown in laboratory culture. The more typical visibly luminous (VL) type of V. fischeri co-occurs in Hawaiian seawater with these NVL strains; thus, two phenotypically distinct groups of this species potentially have access to the symbiotic niche, yet only the NVL ones are found there. In laboratory inoculation experiments, VL strains, when presented in pure culture, showed the same capability for colonizing the light organ as NVL strains. However, in experiments with mixed cultures composed of both VL and NVL strains, the VL ones were unable to compete with the NVL ones and did not persist within the light organ as the symbiosis became established. In addition, NVL strains entered light organs that had already been colonized by VL strains and displaced them. The mechanism underlying the symbiotic competitiveness exhibited by NVL strains remains unknown; however, it does not appear to be due to a higher potential for siderophore activity. While a difference in luminescence phenotype between VL and NVL strains in culture is not likely to be significant in the symbiosis, it has helped identify two distinct groups of V. fischeri that express different colonization capabilities in the squid light organ. This competitive difference provides a useful indication of important traits in light organ colonization.
Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus
Authors: Plamann L , Davis J M , Cantwell B , Mayor J
Published: November 2016

The asg mutants of Myxococcus xanthus are defective in production of extracellular A-signal, which serves as a cell density signal for fruiting-body development. The DNA sequence of asgB, one of the three asg genes, was determined. The deduced amino acid sequence of AsgB contains a DNA-binding helix-turn-helix motif near the C terminus. This putative helix-turn-helix is highly similar to the helix-turn-helix in region 4.2 of major sigma factors, which is the region that recognizes and interacts with -35 sequences of promoters. We propose that AsgB is a transcription factor that binds to DNA sequences similar to the -35 hexamer, TTGACA. Analyses of asgB RNA levels and expression of an asgB-lacZ translational fusion indicate that expression of asgB remains fairly constant during the transition from growth into early development. The mutation within the asgB480 allele was identified as an A-to-G transition that results in a threonine-to-alanine substitution in the predicted protein product. Attempts to replace the wild-type copy of asgB with a null allele failed, indicating that asgB may be essential for growth.
Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections
Authors: Kannenberg E L , Perotto S , Bianciotto V , Rathbun E A , Brewin N J
Published: November 2016

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.