bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose
Authors: Bhat R , Marx A , Galanos C , Conrad R S
Published: November 2016

Lipid A derived from Pseudomonas aeruginosa PAO1 contains a biphosphorylated 1-6-linked glucosamine disaccharide backbone. The reducing glucosamine has an unsubstituted glycosidically linked phosphate at C-1. The nonreducing glucosamine has an ester-bound phosphate at C-4' which is nonstoichiometrically substituted with 4-amino-4-deoxyarabinose. Induction of 4-amino-4-deoxyarabinose was dependent on cultural conditions. No pyrophosphate groups were detected. Acyloxyacyl diesters are formed by esterification of the amide-bound 3-hydroxydodecanoic acid with dodecanoic acid and 2-hydroxydodecanoic acids in an approximate molar ratio of 2:1. Dodecanoic and 3-hydroxydecanoic acids are esterified to positions C-3 and C-3' in the sugar backbone. All hydroxyl groups of the glucosamine disaccharide except C-4 and C-6' are substituted. Lipopolysaccharide chemical analyses measured glucose, rhamnose, heptose, galactosamine, alanine, phosphate, and glucosamine. The proposed lipid A structure differs from previous models. There are significant differences in acyloxyacyl diesters, and the proposed model includes an aminopentose substituent.
Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli
Authors: Misawa N , Nakagawa M , Kobayashi K , Yamano S , Izawa Y , Nakamura K , Harashima K
Published: November 2016

The most important function of carotenoid pigments, especially beta-carotene in higher plants, is to protect organisms against photooxidative damage (G. Britton, in T. W. Goodwin, ed., Plant Pigments--1988, 1988; N. I. Krinsky, in O. Isler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). beta-Carotene also functions as a precursor of vitamin A in mammals (G. A. J. Pitt, in I. Osler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). The enzymes and genes which mediate the biosynthesis of cyclic carotenoids such as beta-carotene are virtually unknown. We have elucidated for the first time the pathway for biosynthesis of these carotenoids at the level of enzyme-catalyzed reactions, using bacterial carotenoid biosynthesis genes. These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined. Six open reading frames were found and designated the crtE, crtX, crtY, crtI, crtB, and crtZ genes in reference to the carotenoid biosynthesis genes of a photosynthetic bacterium, Rhodobacter capsulatus; only crtZ had the opposite orientation from the others. The carotenoid biosynthetic pathway in Erwinia uredovora was clarified by analyzing carotenoids accumulated in E. coli transformants in which some of these six genes were expressed, as follows: geranylgeranyl PPiCrtB----prephytoene PPiCrtE----phytoeneCrtI---- lycopeneCrtY----beta-caroteneCrtZ----zeaxanthinCrtX--- -zeaxanthin-beta- diglucoside. The carotenoids in this pathway appear to be close to those in higher plants rather than to those in bacteria. Also significant is that only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene.
Overproduction of release factor reduces spontaneous frameshifting and frameshift suppression by mutant elongation factor Tu
Authors: Aulin M R , Hughes D
Published: November 2016

Mutant forms of elongation factor Tu encoded by tufA8 and tufB103 in Salmonella typhimurium cause suppression of some but not all frameshift mutations. All of the suppressed mutations in S. typhimurium have frameshift windows ending in the termination codon UGA. Because both tufA8 and tufB103 are moderately efficient UGA suppressors, we asked whether the efficiency of frameshifting is influenced by the level of misreading at UGA. We introduced plasmids synthesizing either one of the release factors into strains in which the tuf mutations suppress a test frameshift mutation. We found that overproduction of release factor 2 (which catalyzes release at UGA and UAA) reduced frameshifting promoted by the tuf mutations at all sites tested. However, at one of these sites, trpE91, overproduction of release factor 1 also reduced suppression. The spontaneous level of frameshift "leakiness" at three sites in trpE, each terminating in UGA, was reduced in strains carrying the release factor 2 plasmid. We conclude that both spontaneous and suppressor-enhanced reading-frame shifts are influenced by the activity of peptide chain release factors. However, the data suggest that the effect of release factor on frameshifting does not necessarily depend on the presence of the normal triplet termination signal.
Comparative genetic organization of incompatibility group P degradative plasmids
Authors: Burlage R S , Bemis L A , Layton A C , Sayler G S , Larimer F
Published: November 2016

Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.
Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis
Authors: Zuberi A R , Ying C W , Parker H M , Ordal G W
Published: November 2016

We have used Tn917lacZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsI. An additional transposon insert that maps in the hag locus was obtained. The pattern of beta-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtilis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response.
Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum
Authors: Gerischer U , Dürre P
Published: November 2016

Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined. Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9. In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity. The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation. From the E. coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme. Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain. The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity. It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced. Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene. Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein. Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence. Together with these regulatory sequences, the adc gene formed a single operon. The carboxyl terminus of the enzyme proved to be rather hydrophobic. In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed. Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis. The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase.
Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones
Authors: Yoshida H , Bogaki M , Nakamura S , Ubukata K , Konno M
Published: November 2016

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.
Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family of site-specific recombinases
Authors: Colloms S D , Sykora P , Szatmari G , Sherratt D J
Published: November 2016

Site-specific recombination at the plasmid ColE1 cer site requires the Escherichia coli chromosomal gene xerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD. The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodes a protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene is expressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames.
Extracellular Ca2(+)-dependent inducible alkaline phosphatase from extremely halophilic archaebacterium Haloarcula marismortui
Authors: Goldman S , Hecht K , Eisenberg H , Mevarech M
Published: November 2016

When starved of inorganic phosphate, the extremely halophilic archaebacterium Haloarcula marismortui produces the enzyme alkaline phosphatase and secretes it to the medium. This inducible extracellular enzyme is a glycoprotein whose subunit molecular mass is 160 kDa, as estimated by sodium dodecyl sulfate-gel electrophoresis. The native form of the enzyme is heterogeneous and composed of multiple oligomeric forms. The enzymatic activity of the halophilic alkaline phosphatase is maximal at pH 8.5, and the enzyme is inhibited by phosphate. Unlike most alkaline phosphatases, the halobacterial enzyme requires Ca2+ and not Zn2+ ions for its activity. Both calcium ions (in the millimolar range) and NaCl (in the molar range) are required for the stability of the enzyme.
Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase
Authors: Elliott T , Avissar Y J , Rhie G E , Beale S I
Published: November 2016

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.
Responses to multiple-nutrient starvation in marine Vibrio sp. strain CCUG 15956
Authors: Nyström T , Flärdh K , Kjelleberg S
Published: November 2016

The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.
Effects of glutamines and glutamates at sites of covalent modification of a methyl-accepting transducer
Authors: Park C , Dutton D P , Hazelbauer G L
Published: November 2016

Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.
Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK
Authors: Totten P A , Lory S
Published: November 2016

Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein. We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P. A. Totten and S. Lory, J. Bacteriol. 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin. The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella. However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P. aeruginosa PAK (45 kDa), indicating degradation in E. coli or modification in P. aeruginosa. Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms. The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E. coli (and Bacillus subtilis). Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene. Models for the involvement of both RpoN and RpoF in the expression of flagellin in P. aeruginosa are presented.
DNA gyrase gyrA mutations in ciprofloxacin-resistant strains of Staphylococcus aureus: close similarity with quinolone resistance mutations in Escherichia coli
Authors: Sreedharan S , Oram M , Jensen B , Peterson L R , Fisher L M
Published: November 2016

The gyrA genes isolated from three ciprofloxacin-resistant clinical isolates of Staphylococcus aureus carried codon 84 (serine----leucine) and/or codon 85 (serine----proline) mutations that were absent in pretreatment susceptible strains. These substitutions occur in a region of the gyrase A protein wherein directly analogous mutations of serine 83----leucine and alanine 84----proline in Escherichia coli confer quinolone resistance. Thus, DNA gyrase A subunit mutations are implicated in resistance to ciprofloxacin in S. aureus.
Lysis protein S of phage lambda functions in Saccharomyces cerevisiae
Authors: Garrett J , Bruno C , Young R
Published: November 2016

The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control. Induction with galactose in S. cerevisiae terminated cell growth and prevented colony formation. Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene. These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.
DNA sequences required for the alkalophily of Bacillus sp. strain C-125 are located close together on its chromosomal DNA
Authors: Kudo T , Hino M , Kitada M , Horikoshi K
Published: November 2016

Two alkali-sensitive mutants of alkalophilic Bacillus sp. strain C-125 were obtained. Mutant 38154 showed defective regulation of internal pH. Plasmids pALK1 and pALK2, containing DNA fragments different from those of the parent strain, were able to recover alkalophily in mutants 18224 and 38154, respectively. DNA analysis suggested that the two fragments overlapped on the chromosomal DNA of strain C-125.
recA and catalase in H2O2-mediated toxicity in Neisseria gonorrhoeae
Authors: Hassett D J , Charniga L , Cohen M S
Published: November 2016

Neisseria gonorrhoeae cells defective in the biosynthesis of the recA gene product are no more sensitive to hydrogen peroxide than wild-type cells. Although gonococci possess nearly 100-fold-greater catalase levels than Escherichia coli, they are more susceptible to hydrogen peroxide than this organism. The natural niche of gonococci undoubtedly results in exposure to oxidant stress; however, they do not demonstrate particularly efficient antioxidant defense systems.
Proteolysis and modulation of the activity of the cell division inhibitor SulA in Escherichia coli lon mutants
Authors: Canceill D , Dervyn E , Huisman O
Published: November 2016

Intracellular accumulation of the inducible cell division inhibitor SulA is modulated by proteases that ensure its degradation, namely, the Lon protease and another ATP-dependent protease(s). Lon- cells are UV sensitive because SulA is stable. We asked whether these ATP-dependent proteases are more active when lon cells are grown at high temperature or in synthetic medium since these conditions decrease the UV sensitivity of lon cells. We found that these growth conditions have no direct effect on Lon-independent degradation of SulA. They may, instead, decrease the SulA-FtsZ interaction.
Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB)
Authors: Uphoff T S , Welch R A
Published: November 2016

We cloned a 13.5-kilobase EcoRI fragment containing the calcium-independent hemolysin determinant (pWPM110) from a clinical isolate of Proteus mirabilis (477-12). The DNA sequence of a 7,191-base-pair region of pWPM110 was determined. Two polypeptides are encoded in this region, HpmB and HpmA (in that transcriptional order), with predicted molecular masses of 63,204 and 165,868 daltons, respectively. A putative Fur-binding site was identified upstream of hpmB overlapping the -35 region of the proposed hpm promoter. In vitro transcription-translation of pWPM110 DNA and other subclones confirmed the assignment of molecular masses for the predicted polypeptides. These polypeptides are predicted to have NH2-terminal leader peptides of 17 and 29 amino acids, respectively. NH2-terminal amino acid sequence analysis of purified extracellular hemolysin (HpmA) confirmed the cleavage of the 29-amino-acid leader peptide in the secreted form of HpmA. Hemolysis assays and immunoblot analysis of Escherichia coli containing subclones expressing hpmA, hpmB, or both indicated that HpmB is necessary for the extracellular secretion and activation of HpmA. Significant nucleotide identity (52.1%) was seen between hpm and the shl hemolysin gene sequences of Serratia marcescens despite differences in the G+C contents of these genes (hpm, 38%; shl, 65%). The predicted amino acid sequences of HpmB and HpmA are also similar to those of ShlB and ShlA, the respective sequence identities being 55.4 and 46.7%. Predicted cysteine residues and major hydrophobic and amphipathic domains have been strongly conserved in both proteins. Thus, we have identified a new hemolysin gene family among gram-negative opportunistic pathogens.
HlyB-dependent secretion of hemolysin by uropathogenic Escherichia coli requires conserved sequences flanking the chromosomal hly determinant
Authors: Cross M A , Koronakis V , Stanley P L , Hughes C
Published: November 2016

The synthesis and secretion of hemolysin (HlyA) by Escherichia coli are governed by four contiguous genes (hlyCABD) that are closely conserved on plasmids and, among human pathogenic strains, on the chromosome. We have previously shown that in plasmid pHly152 the coexpressed synthesis and export functions are uncoupled by intraoperon transcription termination, which is in turn alleviated by antitermination dictated in cis by a region upstream of the hly operon. In this study we describe an analogous region of ca. 1,100 base pairs flanking the chromosomal hly determinant of the uropathogenic strain E. coli 2001. This region had no significant effect on intracellular levels of hemolysin but activated strongly, both in cis and in trans, the specific hlyB-hlyD-dependent hemolysin secretion function. The secretion-activating region increased the transcription of the secretion gene hlyB, but the transcription effect was not as pronounced as that seen in the pHly152 determinant and was not evident when the region was present in trans to the hemolysin genes, suggesting that, in addition to transcriptional activation, the region may possibly exert a secondary posttranscriptional influence. Southern hybridizations with the 1,100-base pairs secretion-activating sequence showed low identity to plasmid pHly152 and no identity with total DNA from nonhemolytic uropathogenic E. coli or hemolytic isolates of Proteus vulgaris, Proteus mirabilis, and Morganella morganii. In contrast, hybridization to total DNA from hemolytic E. coli isolates belonging to different serotypes showed strong conservation of the activating sequence, indicating that it is an integral component of the chromosomal hly determinant that is widespread among uropathogenic E. coli.
Enhancement of protein translocation across the membrane by specific mutations in the hydrophobic region of the signal peptide
Authors: Goldstein J , Lehnhardt S , Inouye M
Published: November 2016

The hydrophobic region of the signal peptide of the OmpA protein of the Escherichia coli outer membrane was extensively altered in its hydrophobicity and predicted secondary structure by site-specific mutagenesis. The mutated signal peptides were fused to nuclease A from Staphylococcus aureus, and the function of the signal peptide was examined by measuring the rate of processing of the signal peptide. Six of the 12 mutated signal peptides in the nuclease hybrid were processed faster than the wild-type. In particular, the processing of the mutated signal peptide in which the alanine residue at position 9 was substituted with a valine residue was enhanced almost twofold over the processing of the wild-type signal peptide. In addition, the production of nuclease A fused with this mutated signal peptide also increased twofold. However, these effects were not observed when the mutated signal peptide was fused to TEM beta-lactamase. Analysis of the present mutations suggests that both overall hydrophobicity and distinct structural requirements in the hydrophobic region have important roles in signal peptide function.
Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans
Authors: Rustchenko-Bulgac E P , Sherman F , Hicks J B
Published: November 2016

At frequencies as high as 1.4%, the pathogenic yeast Candida albicans spontaneously gave rise to morphological mutants exhibiting more than 20 different types of abnormal colonies; approximately two-thirds of the mutants were stable, while the other one-third were unstable and produced mixtures of different colonial forms at very high rates. Abnormal electrophoretic karyotypes were observed for all of the 14 mutants that were examined, indicating that they were associated with different types of single and multiple gross chromosomal rearrangements. Because C. albicans is asexual and does not go through a meiotic cycle, we suggest that the high frequency of chromosomal rearrangements provides a means for genetic variation in this organism.
Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants
Authors: Jones C J , Macnab R M
Published: November 2016

The process of flagellar assembly in Salmonella typhimurium was investigated by using temperature-sensitive mutants. The mutants were grown at the restrictive temperature and then at the permissive temperature, with radiolabel supplied in the first phase of the experiment and not the second, or vice versa. Flagellar hook-basal body complexes were then purified and analyzed by gel electrophoresis and autoradiography. The extent to which a given protein was labeled in the two phases of the experiment provided information as to whether it preceded or followed the block caused by the mutant protein. We conclude the following concerning flagellar assembly. The M-ring protein (FliF) is stably incorporated in the earliest stage detected, along with two previously unknown proteins, with apparent molecular masses of 23 and 26 kilodaltons, respectively, and possibly one of the switch components, FliG. Independent of that event and all other events, the P-ring and L-ring proteins (FlgI and FlgH) are synthesized and exported to the periplasm and outer membrane by the primary cellular export pathway. Rod assembly occurs by export (via the flagellum-specific pathway) of subunits of four proteins, FlgB, FlgC, FlgF, and FlgG, and their incorporation, probably in that order, into the rod structure; this stage requires the flhA and fliI genes, perhaps because they encode part of the export apparatus. Once rod assembly is complete, the FlgI and FlgH proteins assemble around the rod to form the P and L rings. The rod structure, which is only metastable while it is being constructed, becomes stable upon P-ring addition. Export (via the flagellum-specific pathway) and assembly of hook protein, hook-associated proteins, and filament protein then occur successively. A number of flagellar proteins, whose genetic origin and structural role are not yet known, were identified on the basis of their dependence on the flagellar master operon for expression.
Enzymes of ammonia assimilation in hyphae and vesicles of Frankia sp. strain CpI1
Authors: Schultz N A , Benson D R
Published: November 2016

Frankia spp. are filamentous actinomycetes that fix N2 in culture and in actinorhizal root nodules. In combined nitrogen-depleted aerobic environments, nitrogenase is restricted to thick-walled spherical structures, Frankia vesicles, that are formed on short stalks along the vegetative hyphae. The activities of the NH4(+)-assimilating enzymes (glutamine synthetase [GS], glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase) were determined in cells grown on NH4+ and N2 and in vesicles and hyphae from N2-fixing cultures separated on sucrose gradients. The two frankial GSs, GSI and GSII, were present in vesicles at levels similar to those detected in vegetative hyphae from N2-fixing cultures as shown by enzyme assay and two-dimensional polyacrylamide gel electrophoresis. Glutamate synthase, glutamate dehydrogenase, and alanine dehydrogenase activities were restricted to the vegetative hyphae. Vesicles apparently lack a complete pathway for assimilating ammonia beyond the glutamine stage.
Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants
Authors: Dylan T , Nagpal P , Helinski D R , Ditta G S
Published: November 2016

Nodule development (ndv) mutants of Rhizobium meliloti cannot invade alfalfa to establish a nitrogen-fixing symbiosis and instead induce the formation of small, white, unoccupied nodules on alfalfa roots. Such mutants also fail to produce the unusual cyclic oligosaccharide beta-(1----2)-glucan and show defects in several aspects of vegetative growth and function. Here we show that ndv mutants are severely reduced, although not totally deficient, in the ability to attach to and initiate infection threads on alfalfa seedlings, and we demonstrate that the symbiotic deficiency can be separated from the rest of the mutant phenotype by isolating second-site pseudorevertants. Pseudorevertants selected for restoration of motility, a vegetative property, regained a substantial amount of attachment capability but only slight infection thread initiation and symbiotic ability. Such strains also regained partial tolerance to growth at low osmolarity, even though they did not recover the ability to synthesize periplasmic beta-(1----2)-glucan. Pseudorevertants selected on alfalfa for restoration of symbiosis were unrestored for beta-(1----2)-glucan production or any other vegetative property and regained little or no attachment or infection thread initiation capability. We take these data to indicate that wild-type R. meliloti normally has considerable excess capability for both attachment and infection thread initiation and that the symbiotic block in ndv mutants lies further along the developmental pathway than either of these processes, probably at the level of infection thread extension. Further, the fact that neither type of pseudorevertant recovered the ability to produce periplasmic beta-(1----2)-glucan raises the possibility that this oligosaccharide is not directly required for nodule development.
Permeability barrier to hydrophilic solutes in Mycobacterium chelonei
Authors: Jarlier V , Nikaido H
Published: November 2016

In order to define the permeability barrier to hydrophilic molecules in mycobacteria, we used as a model a smooth, beta-lactamase-producing strain of Mycobacterium chelonei. The rates of hydrolysis of eight cephalosporins by intact and sonicated cells were measured, and the permeability coefficient (P) was calculated from these rates by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). P ranged from (0.9 +/- 0.3) x 10(-8) (benzothienylcephalosporin) to (10 +/- 3.3) x 10(-8) cm/s (cephaloridine); i.e., the P values were lower than those reported for Pseudomonas aeruginosa and Escherichia coli by 1 and 3 orders of magnitude, respectively. The permeability barrier was shown to reduce drastically the stream of drug molecules entering the cell, allowing the rather low level of beta-lactamase (0.1 U/mg of protein with penicillin G) to decrease radically the concentration of the drug at the target; this explains the poor in vitro activities of the beta-lactams against M. chelonei. We also estimated P for small, hydrophilic molecules (glucose, glycerol, glycine, leucine), by studying their uptake kinetics. The values found, ranging from 15 x 10(-8) to 490 x 10(-8) cm/s, were consistent again with a very low permeability of M. chelonei cell wall. The permeation of cephalosporins was not very dependent on the hydrophobicity of the molecules or on the temperature, suggesting a hydrophilic pathway of penetration for these molecules.
Chloride transport pathways and their bioenergetic implications in the obligate acidophile Bacillus coagulans
Authors: McLaggan D , Keyhan M , Matin A
Published: November 2016

The protonophore-mediated collapse of the large delta pH that acidophiles maintain across their cytoplasmic membranes was augmented by the presence of Cl-, and Cl- influx into the cells occurred evidently in response to the protonophore-induced increase in the inside-positive membrane potential (+ delta psi). In respiring cells, the addition of Cl- but not SO4(2-) salts caused a rapid and precipitous decrease in the + delta psi. A Nernstian relationship between the imposed transmembrane K+ gradient and the valinomycin-induced K+ diffusion potentials was observed when everted membrane vesicles were loaded with K2SO4 or KH2PO4 but not when loaded with KCl or KNO3. Thus, electrogenic Cl- transport occurred in Bacillus coagulans. In addition, a nonelectrogenic temperature-sensitive Cl- transport mechanism, with the net Cl- efflux coefficient (PCl-) ranging from 1.5 x 10(-4) to 6.1 x 10(-6) cm/s, accounted for the massive Cl- efflux from Cl(-)-loaded cells. Thus, B. coagulans, despite its dependence on the + delta psi and therefore the need to exclude anions, apparently possesses specific mechanisms for Cl- permeation. Active cells of B. coagulans prevented Cl- accumulation from attaining an electrochemical equilibrium, maintaining a delta micro Cl- of ca. -63 mV. B. coagulans therefore also possesses an energy-dependent mechanism for Cl- exclusion from the cells.
Identification of plant-inducible genes in Erwinia chrysanthemi 3937
Authors: Beaulieu C , Van Gijsegem F
Published: November 2016

We present a method for identifying plant-inducible genes of Erwinia chrysanthemi 3937. Mutagenesis was done with the Mu dIIPR3 transposon, which carries a promoterless neomycin phosphotransferase gene (nptI), so upon insertion, the truncated gene can fuse to E. chrysanthemi promoters. Mutants containing insertions in plant-inducible genes were selected for their sensitivity to kanamycin on minimal plates and for their acquired resistance to this antibiotic when an S. ionantha plant extract was added to kanamycin minimal plates. The selection allowed the identification of E. chrysanthemi promoters inducible by host factors present in the S. ionantha plant extract. Using this method, we isolated 30 mutants and characterized 10 of them. Two mutants were defective in cation uptake, one was defective in the galacturonate degradation pathway, and another was altered in the production of the acidic pectate lyase. The functions of the other mutated genes are still unknown, but we show that most of them are involved in pathogenicity.
Possible involvement of the division cycle in dispersal of Escherichia coli from biofilms
Authors: Allison D G , Evans D J , Brown M R , Gilbert P
Published: November 2016

Growth rate control of adherent, sessile populations was achieved by the controlled perfusion of membrane-associated bacterial biofilms by the method of Gilbert et al. (P. Gilbert, D. G. Allison, D. J. Evans, P. S. Handley, and M. R. W. Brown, Appl. Environ. Microbiol. 55:1308-1311, 1989). Changes in cell surface hydrophobicity were evaluated with respect to growth rate for such sessile Escherichia coli cells and compared with those of suspended (planktonic) populations grown in a chemostat. Newly formed daughter cells shed at the various growth rates from the biofilm during its growth and development were also included in the study. Surface hydrophobicity decreased with growth rate similarly for both planktonic and sessile E. coli; no significant differences were noted between the two. Daughter cells dislodged from the biofilm, however, were significantly more hydrophilic than those remaining, indicating that hydrophobicity changed during the division cycle. Our data support the hypothesis that dispersal of cells from adhesive biofilms and recolonization of new surfaces reflect cell-cycle-mediated events.
Integration of bacteriophage lambda into the cryptic lambdoid prophages of Escherichia coli
Authors: Lichens-Park A , Smith C L , Syvanen M
Published: November 2016

Bacteriophage lambda missing its chromosomal attachment site will integrate into recA+ Escherichia coli K-12 and C at the sites of cryptic prophages. The specific regions in which these recombination events occur were identified in both lambda and the bacterial chromosomes. A NotI restriction site on the prophage allowed its physical mapping. This allowed us to identify the locations of Rac, Qin, and Qsr' cryptic prophages on the NotI map of E. coli K-12 and, by analogy, to identify the cryptic prophage in E. coli C as Qin. No new cryptic prophages were detected in E. coli K-12.
Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer
Authors: Komano T , Funayama N , Kim S R , Nisioka T
Published: November 2016

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.
Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity
Authors: Peters J E , Galloway D R
Published: November 2016

A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P.A. Schad and B.H. Iglewski, J. Bacteriol. 170:2784-2789, 1988). However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.
Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4
Authors: Kaphammer B , Kukor J J , Olsen R H
Published: November 2016

The closely linked structural genes tfdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, tfdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Derepression of tfdC was achieved when Pseudomonas aeruginosa PAO4032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.
Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon
Authors: Owolabi J B , Rosen B P
Published: November 2016

The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals. The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC. The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities. Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription. The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb). This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein. Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes. In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein.