bluuebunny/crossref_metadata_2025 huggingface dataset:
The Bradyrhizobium japonicum fegA gene encodes an iron-regulated outer membrane protein with similarity to hydroxamate-type siderophore receptors
Authors: LeVier K , Guerinot M L
Published: November 2016

Iron is important in the symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, yet little is known about rhizobial iron acquisition strategies. Analysis of outer membrane proteins (OMPs) from B. japonicum 61A152 identified three iron-regulated OMPs in the size range of several known receptors for Fe(III)-scavenging siderophores. One of the iron-regulated proteins, FegA, was purified and microsequenced, and a reverse genetics approach was used to clone a fegA-containing DNA fragment. Sequencing of this fragment revealed a single open reading frame of 750 amino acids. A putative N-terminal signal sequence of 14 amino acids which would result in a mature protein of 736 amino acids with a molecular mass of 80,851 Da was predicted. FegA shares significant amino acid similarity with several Fe(III)-siderophore receptors from gram-negative bacteria and has greater than 50% amino acid similarity and 33% amino acid identity with two [corrected] bacterial receptors for hydroxamate-type Fe(III)-siderophores. A dendrogram describing total inferred sequence similarity among 36 TonB-dependent OMPs was constructed; FegA grouped with Fe(III)-hydroxamate receptors. The transcriptional start site of fegA was mapped by primer extension analysis, and a putative Fur-binding site was found in the promoter. Primer extension and RNA slot blot analysis demonstrated that fegA was expressed only in cells grown under iron-limiting conditions. This is the first report of the cloning of a gene encoding a putative Fe(III)-siderophore receptor from nitrogen-fixing rhizobia.
Interactions of Escherichia coli UmuD with activated RecA analyzed by cross-linking UmuD monocysteine derivatives
Authors: Lee M H , Walker G C
Published: November 2016

SOS mutagenesis in Escherichia coli requires the participation of a specialized system involving the activated form of UmuD (UmuD'), UmuC, RecA, and DNA polymerase III proteins. We have used a set of monocysteine derivatives of UmuD (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994) and the cysteine-specific photoactive cross-linker p-azidoiodoacetanilide (AIA) to study not only the interactions of intact UmuD in the homodimer but also the interactions of UmuD with activated RecA. The reactivities of the individual UmuD monocysteine derivatives with AIA were similar to their reactivities with iodoacetate. The relative efficiencies of cross-linking of the AIA-modified monocysteine UmuD derivatives in the homodimer form are also consistent with our previous conclusions concerning the relative closeness of various UmuD residues to the dimer interface. With respect to the UmuD-RecA interface, the AIA-modified VC34 and SC81 monocysteine derivatives cross-linked most efficiently with RecA, indicating that positions 34 and 81 of UmuD are closer to the RecA interface than the other positions we tested. The AIA-modified SC57, SC67, and SC112 monocysteine derivatives cross-linked moderately efficiently with RecA. Neither C24, the wild-type UmuD that has a cysteine located at the Cys-24-Gly-25 cleavage site, nor SC60, the UmuD monocysteine derivative with a cysteine substitution at the position of the putative active-site residue, was able to cross-link with RecA, suggesting that RecA need not directly interact with residues involved in the cleavage reaction. SC19, located in the N-terminal fragment of UmuD that is cleaved, and LC44 also did not cross-link efficiently with RecA.
Inhibition of RecA-mediated cleavage in covalent dimers of UmuD
Authors: Lee M H , Guzzo A , Walker G C
Published: November 2016

Disulfide-cross-linked UmuD2 derivatives were cleaved poorly upon incubation with activated RecA. Reducing the disulfide bonds prior to incubating the derivatives with RecA dramatically increased their extent of cleavage. These observations suggest that the UmuD monomer is a better substrate for the RecA-mediated cleavage reaction than the dimer.
Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN
Authors: Callahan C , Deutscher M P
Published: November 2016

The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN. Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth. On the basis of these data, we suggest that o290 be renamed rbn. Potential homologs of rbn in other organisms also were identified.
Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae
Authors: McBride M J , Kempf M J
Published: November 2016

Cytophaga johnsonae displays many features that make it an excellent model of bacterial gliding motility. Unfortunately, genetic analyses of C. johnsonae, or any related gliding bacteria, were not possible because of a complete lack of selectable markers, cloning vectors, transposons, and convenient methods of gene transfer. As a first step toward a molecular analysis of gliding motility of C. johnsonae, we developed these genetic techniques and tools. Common broad-host-range plasmids and transposons did not function in C. johnsonae. We identified one Bacteroides transposon, Tn4351, that could be introduced into C. johnsonae on plasmid R751 by conjugation from Escherichia coli. Tn4351 inserted in the C. johnsonae genome and conferred erythromycin resistance. Tn-4351 insertions resulted in auxotrophic mutations and motility mutations. We constructed novel plasmids and cosmids for genetic analyses of C. johnsonae. These cloning vectors are derived from a small cryptic plasmid (pCP1) that we identified in the fish pathogen Cytophaga psychrophila D12. These plasmids contain the ermF (erythromycin resistance) gene from Tn4351 and a variety of features that facilitate propagation and selection in E. coli and conjugative transfer from E. coli to C. johnsonae.
Alginate synthesis in Pseudomonas aeruginosa: the role of AlgL (alginate lyase) and AlgX
Authors: Monday S R , Schiller N L
Published: November 2016

Previous studies localized an alginate lyase gene (algL) within the alginate biosynthetic gene cluster at 34 min on the Pseudomonas aeruginosa chromosome. Insertion of a Tn501 polar transposon in a gene (algX) directly upstream of algL in mucoid P. aeruginosa FRD1 inactivated expression of algX, algL, and other downstream genes, including algA. This strain is phenotypically nonmucoid; however, alginate production could be restored by complementation in trans with a plasmid carrying all of the genes inactivated by the insertion, including algL and algX. Alginate production was also recovered when a merodiploid that generated a complete alginate gene cluster on the chromosome was constructed. However, alginate production by merodiploids formed in the algX::Tn501 mutant using an alginate cluster with an algL deletion was not restored to wild-type levels unless algL was provided on a plasmid in trans. In addition, complementation studies of Tn501 mutants using plasmids containing specific deletions in either algL or algX revealed that both genes were required to restore the mucoid phenotype. Escherichia coli strains which expressed algX produced a unique protein of approximately 53 kDa, consistent with the gene product predicted from the DNA sequencing data. These studies demonstrate that AlgX, whose biochemical function remains to be defined, and AlgL, which has alginate lyase activity, are both involved in alginate production by P. aeruginosa.
Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae
Authors: Collinson S K , Clouthier S C , Doran J L , Banser P A , Kay W W
Published: November 2016

Salmonella enteritidis produces thin, aggregative fimbriae, named SEF17, which are composed of polymerized AgfA fimbrin proteins. DNA sequence analysis of a 2-kb region of S. enteritidis DNA revealed three contiguous genes, agfBAC. The 453-bp agfA gene encodes the AgfA fimbrin, which was predicted to be 74% identical and 86% similar in primary sequence to the Escherichia coli curli structural protein, CsgA. pHAG, a pUC18 derivative containing a 3.0-kb HindIII fragment encoding agfBAC, directed the in vitro expression of the major AgfA fimbrin, with an M(r) of 17,000, and a minor AgfB protein, with an M(r) of 16,000, encoded by the 453-bp agfB gene. AgfA was not expressed from pDAG, a pUC18 derivative containing a 3.1-kb DraI DNA fragment encoding agfA but not agfB. Primer extension analysis identified two adjacent transcription start sites located immediately upstream of agfB in positions analogous to those of the E. coli curlin csgBA operon. No transcription start sites were located immediately upstream of agfA or agfC. Northern (RNA) blot analysis confirmed that transcription of agfA was initiated from the agfB promoter region. Secondary-structure analysis of the putative mRNA transcript for agfBAC predicted the formation of a stem-loop structure (delta Gzero, -22 kcal/mol [-91 kJ/mol]) in the intercistronic region between agfA and agfC, which may be involved in stabilization of the agfBA portion of the agfBAC transcript. agfBAC and flanking regions had a high degree of sequence similarity with those counterparts of the E. coli curlin csgBA region for which sequence data are available. These data are demonstrative of the high degree of similarity between S. enteritidis SEF17 fimbriae and E. coli curli with respect to fimbrin amino acid sequence and genetic organization and, therefore, are indicative of a common and relatively recent ancestry.
Mutants of Myxococcus xanthus dsp defective in fibril binding
Authors: Chang B Y , Dworkin M
Published: November 2016

The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils is not necessary for group motility, myxospore formation, or the early aggregative stage of development. We propose, however, that the perception of fibril binding is required for normal social behavior and development. The dsp fbd mutants (from here on referred to as fbd mutants) open the possibility of isolating and characterizing a putative fibril receptor gene.
Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli
Authors: Raux E , Lanois A , Levillayer F , Warren M J , Brody E , Rambach A , Thermes C
Published: November 2016

In order to study the Salmonella typhimurium cobalamin biosynthetic pathway, the S. typhimurium cob operon was isolated and cloned into Escherichia coli. This approach has given the new host of the cob operon the ability to make cobalamins de novo, an ability that had probably been lost by this organism. In total, 20 genes of the S. typhimurium cob operon have been transferred into E. coli, and the resulting recombinant strains have been shown to produce up to 100 times more corrin than the parent S. typhimurium strain. These measurements have been performed with a quantitative cobalamin microbiological assay which is detailed in this work. As with S. typhimurium, cobalamin synthesis is only observed in the E. coli cobalamin-producing strains when they are grown under anaerobic conditions. Derivatives of the cobalamin-producing E. coli strains were constructed in which genes of the cob operon were inactivated. These strains, together with S. typhimurium cob mutants, have permitted the determination of the genes necessary for cobalamin production and classification of cbiD and cbiG as cobl genes. When grown in the absence of endogenous cobalt, the oxidized forms of precorrin-2 and precorrin-3, factor II and factor III, respectively, were found to accumulate in the cytosol of the corrin-producing E. coli. Together with the finding that S. typhimurium cbiL mutants are not complemented with the homologous Pseudomonas denitrificans gene, these results lend further credence to the theory that cobalt is required at an early stage in the biosynthesis of cobalamins in S. typhimurium.
Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans
Authors: Ueda S , Yabutani T , Maehara A , Yamane T
Published: November 2016

A 3.6-kb EcoRI-SalI fragment of Paracoccus denitrificans DNA hybridized with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthase gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis of this region showed the presence of a 1,872-bp open reading frame (ORF), which corresponded to a polypeptide with a molecular weight of 69,537. Upstream of the ORF, a promoter-like sequence was found. Escherichia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of beta-ketothiolase and acetoacetylcoenzyme A reductase genes of A. eutrophus. The ORF was designated phaCPd. A plasmid vector carrying the phaCPd'-'lacZ fusion gene downstream of the promoter-like sequence expressed beta-galactosidase activity in P. denitrificans. When a multicopy and broad-host-range vector carrying the ORF along with the promoter-like sequence was introduced into P. denitrificans, the PHA content in the cells increased by twofold compared with cells carrying only a vector sequence.
Expression from the nifB promoter of Azotobacter vinelandii can be activated by NifA, VnfA, or AnfA transcriptional activators
Authors: Drummond M , Walmsley J , Kennedy C
Published: November 2016

In Azotobacter vinelandii, nifB is required for the activity of all three nitrogenases. Expression of a nifB-lacZ fusion was examined to determine which regulatory gene products are important for nifB expression and how its transcription is regulated in response to metals. In all conditions, expression in A. vinelandii was eliminated by an rpoN mutation, confirming the absolute requirement for sigma N. In the wild type, nifB-lacZ expression was approximately twofold higher in cells grown with Mo than without. Expression was negligible in a nifA mutant grown with Mo but was much higher in Mo-free medium, suggesting that in these conditions, another sigma N-dependent activator was responsible for nifB expression, possibly VnfA, AnfA, or NtrC. Although expression of the nifB-lacZ fusion in A. vinelandii vnfA, anfA, and ntrC mutants was little different from that in the wild type, nifB transcription could be activated by NifA, VnfA, or a truncated form of AnfA in Escherichia coli. The two potential NifA binding sites centered at -87 and -129 bp upstream of the transcription start site each overlapped a VnfA recognition sequence, motifs also found in Azotobacter chroococcum in two exactly conserved regions. Deletion analysis showed that both regions are important for nifB expression. Activation of the full-length promoter by AnfA was impaired by overexpressing the DNA-binding domain of NifA, suggesting that binding of NifA and AnfA can be competitive.
Fine tangled pili expressed by Haemophilus ducreyi are a novel class of pili
Authors: Brentjens R J , Ketterer M , Apicella M A , Spinola S M
Published: November 2016

Haemophilus ducreyi synthesizes fine, tangled pili composed predominantly of a protein whose apparent molecular weight is 24,000 (24K). A hybridoma, 2D8, produced a monoclonal antibody (MAb) that bound to a 24K protein in H. ducreyi strains isolated from diverse geographic locations. A lambda gt11 H. ducreyi library was screened with MAb 2D8. A 3.5-kb chromosomal insert from one reactive plaque was amplified and ligated into the pCRII vector. The recombinant plasmid, designated pHD24, expressed a 24K protein in Escherichia coli INV alpha F that bound MAb 2D8. The coding sequence of the 24K gene was localized by exonuclease III digestion. The insert contained a 570-bp open reading frame, designated ftpA (fine, tangled pili). Translation of ftpA predicted a polypeptide with a molecular weight of 21.1K. The predicted N-terminal amino acid sequence of the polypeptide encoded by ftpA was identical to the N-terminal amino acid sequence of purified pilin and lacked a cleavable signal sequence. Primer extension analysis of ftpA confirmed the lack of a leader peptide. The predicted amino acid sequence lacked homology to known pilin sequences but shared homology with the sequences of E. coli Dps and Treponema pallidum antigen TpF1 or 4D, proteins which associate to form ordered rings. An isogenic pilin mutant, H. ducreyi 35000ftpA::mTn3(Cm), was constructed by shuttle mutagenesis and did not contain pili when examined by electron microscopy. We conclude that H. ducreyi synthesizes fine, tangled pili that are composed of a unique major subunit, which may be exported by a signal sequence independent mechanism.
Gene integration in the Escherichia coli chromosome mediated by Tn21 integrase (Int21)
Authors: Francia M V , García Lobo J M
Published: November 2016

A replication-thermosensitive, pSC101-derived plasmid containing the int gene and RHS-2 from the integron in Tn21 and a kanamycin resistance marker has been constructed and used to obtain Tn21 integrase (Int21)-mediated plasmid integration in the Escherichia coli chromosome. Colonies carrying an integrated plasmid were obtained after growth at 42 degrees C. Southern hybridization and PCR experiments indicated that they contained the plasmid specifically integrated through the RHS into different positions in the E. coli chromosome. Nucleotide sequence determination of the plasmid-chromosome junctions showed that integration sites in the chromosome were pentanucleotides with the sequence described for Int21 secondary sites.
Isolation and characterization of flagella and flagellin proteins from the Thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae
Authors: Faguy D M , Bayley D P , Kostyukova A S , Thomas N A , Jarrell K F
Published: November 2016

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.
Efficient insertional mutagenesis in lactococci and other gram-positive bacteria
Authors: Maguin E , Prévost H , Ehrlich S D , Gruss A
Published: November 2016

In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.
Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl-acyl carrier protein desaturase and ferredoxin
Authors: Cahoon E B , Mills L A , Shanklin J
Published: November 2016

Expression of a plant delta 6-palmitoyl (16:0)-acyl carrier protein desaturase in Escherichia coli resulted in the accumulation of the novel monounsaturated fatty acids delta 6-hexadecenoic acid (16:1 delta 6) and delta 8-octadecenoic acid. Amounts of 16:1 delta 6 accumulated by E. coli were increased more than twofold by the expression of a plant ferredoxin together with the delta 6-16:0-acyl carrier protein desaturase.
Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system
Authors: Sahm K , Matuschek M , Müller H , Mitchell W J , Bahl H
Published: November 2016

A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and catabolite repression by glucose were identified by sequence analysis and comparison. The biochemical characterization of maltose uptake in T. thermosulfurigenes EM1 revealed two transport systems with Km values of 7 microM (high affinity) and 400 microM (low affinity). We conclude that the high-affinity system, which is specific for maltose and maltotriose, is a binding-protein-dependent transporter encoded by amyEDC. The gene for the putative ATP-binding protein has not yet been identified, and in contrast to similar systems in other bacteria, it is not located in the immediate vicinity of the chromosome.
Three functions of bacteriophage P1 involved in cell lysis
Authors: Schmidt C , Velleman M , Arber W
Published: November 2016

Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA.
Effect of the surface composition of motile Escherichia coli and motile Salmonella species on the direction of galvanotaxis
Authors: Shi W , Stocker B A , Adler J
Published: November 2016

We have reported that motile Escherichia coli K-12 placed in an electric field swims toward the anode but that motile Salmonella typhimurium strains swim toward the cathode, a phenomenon called galvanotaxis (J. Adler and W. Shi, Cold Spring Harbor Symp. Quant. Biol. 53:23-25, 1988). In the present study, we isolated mutants with an altered direction of galvanotaxis. By further analyses of these mutants and by examination of E. coli and Salmonella strains with altered cell surface structure, we have now established a correlation between the direction of galvanotaxis and the surface structure of the cell: motile rough bacteria (that is, those without O polysaccharide; for example, E. coli K-12 and S. typhimurium mutants of classes galE and rfa) swam toward the anode, whereas motile smooth bacteria (that is, those with O polysaccharide; for example, wild-type S. typhimurium LT2) swam toward the cathode. However, smooth bacteria with acidic polysaccharide capsules (K1 in E. coli and Vi in Salmonella typhi) swam toward the anode. Measurements of passive electrophoretic mobility of strains representative of each set were made. We propose that the different directions of galvanotaxis of rough (or capsulate) bacteria and of smooth bacteria are explicable if the negative electrophoretic mobility of flagellar filaments is less than that of rough bodies but greater than that of smooth bodies.
Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA
Authors: Bass S , Gu Q , Christen A
Published: November 2016

We have isolated three multicopy suppressors of the conditional lethal phenotype of a prc (tsp) null strain of Escherichia coli. One of these suppressors included two novel putative protease genes in tandem that map to 3400 kb or 72.5 centisomes on the chromosome. We propose the names hhoA and hhoB, for htrA homolog, to denote that these genes encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease and 36% identical to each other. The HhoA and HhoB proteins are predicted to be 455 and 355 amino acids, respectively, in length. The mature HhoA protein is periplasmic in location, and amino-terminal sequencing shows that it arises following cleavage of a 27-amino-acid signal peptide. Searches of the protein and DNA databases reveal a rapidly growing family of homologous genes in a variety of other bacteria, including several which are required for virulence in their host. Deletion of the hhoAB genes shows that they are not required for viability at high temperatures like the homologous htrA but grow more slowly than wild-type strains. A second multicopy prc suppressor is the dksA (dnaK suppressor) gene, which is also a multicopy suppressor of defects in the heat shock genes dnaK, dnaJ, and grpE. The dksA gene was independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. A third dosage-dependent prc suppressor includes a truncated rare lipoprotein A (rlpA) gene.
Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria
Authors: Pelicic V , Reyrat J M , Gicquel B
Published: November 2016

Expression in mycobacteria of the structural gene sacB, which encodes the Bacillus subtilis levansucrase, was investigated. sacB expression is lethal to Mycobacterium smegmatis and Mycobacterium bovis BCG in the presence of 10% sucrose. sacB could thus be used as a counterselectable marker in mycobacteria.
Osmotic shock induction of capsule synthesis in Escherichia coli K-12
Authors: Sledjeski D D , Gottesman S
Published: November 2016

The genes (cps) involved in the synthesis of the colanic acid capsular polysaccharide in Escherichia coli K-12 are transcriptionally regulated by numerous proteins. Two of these, RcsB and RcsC, share homology with two-component regulatory elements that respond to environmental stimuli. Osmotic shock by sucrose or NaCl transiently increased transcription of a cpsB::lacZ fusion. RcsC and RcsB were essential for osmotic induction of colanic acid synthesis. In contrast to observations in some other osmotically regulated systems, addition of glycine betaine enhanced the osmotic induction of cps::lacZ by both sucrose and NaCl but had no effect alone.
Assembly-defective OmpC mutants of Escherichia coli K-12
Authors: Xiong X , Deeter J N , Misra R
Published: November 2016

Novel ompC(Dex) alleles were utilized to isolate mutants defective in OmpC biogenesis. These ompC(Dex) alleles also conferred sensitivity to sodium dodecyl sulfate (SDS), which permitted the isolation of SDS-resistant and OmpC-specific phage-resistant mutants that remained Dex+. Many mutants acquired resistance against these lethal agents by lowering the OmpC level present in the outer membrane. In the majority of these mutants, a defect in the assembly (metastable to stable trimer formation) was responsible for lowering OmpC levels. The assembly defects in various mutant OmpC proteins were caused by single-amino-acid substitutions involving the G-39, G-42, G-223, G-224, Q-240, G-251, and G-282 residues of the mature protein. This assembly defect was correctable by an assembly suppressor allele, asmA3. In addition, we investigated one novel OmpC mutant in which an assembly defect was caused by a disulfide bond formation between two nonnative cysteine residues. The assembly defect was fully corrected in a genetic background in which the cell's ability to form disulfide bonds was compromised. The assembly defect of the two-cysteine OmpC protein was also mended by asmA3, whose suppressive effect was not achieved by preventing disulfide bond formation in the mutant OmpC protein.
Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin
Authors: Margolin W , Wang R , Kumar M
Published: November 2016

We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.
p-Cumate catabolic pathway in Pseudomonas putida Fl: cloning and characterization of DNA carrying the cmt operon
Authors: Eaton R W
Published: November 2016

Pseudomonas putida F1 utilizes p-cumate (p-isopropylbenzoate) as a growth substrate by means of an eight-step catabolic pathway. A 35.75-kb DNA segment, within which the cmt operon encoding the catabolism of p-cumate is located, was cloned as four separate overlapping restriction fragments and mapped with restriction endonucleases. By examining enzyme activities in recombinant bacteria carrying these fragments and sub-cloned fragments, genes encoding most of the enzymes of the p-cumate pathway were located. Subsequent sequence analysis of 11,260 bp gave precise locations of the 12 genes of the cmt operon. The first three genes, cmtAaAbAc, and the sixth gene, cmtAd, encode the components of p-cumate 2,3-dioxygenase (ferredoxin reductase, large subunit of the terminal dioxygenase, small subunit of the terminal dioxygenase, and ferredoxin, respectively); these genes are separated by cmtC, which encodes 2,3-dihydroxy-p-cumate 3,4-dioxygenase, and cmtB, coding for 2,3-dihydroxy-2,3-dihydro-p-cumate dehydrogenase. The ring cleavage product, 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate, is acted on by a decarboxylase encoded by the seventh gene, cmtD, which is followed by a large open reading frame, cmtI, of unknown function. The next four genes, cmtEFHG, encode 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase, 2-hydroxypenta-2,4-dienoate hydratase, 4-hydroxy-2-oxovalerate aldolase, and acetaldehyde dehydrogenase, respectively, which transform the decarboxylation product to amphibolic intermediates. The deduced amino acid sequences of all the cmt gene products except CmtD and CmtI have a recognizable but low level of identity with amino acid sequences of enzymes catalyzing analogous reactions in other catabolic pathways. This identity is highest for the last two enzymes of the pathway (4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase [acylating]), which have identities of 66 to 77% with the corresponding enzymes from other aromatic meta-cleavage pathways. Recombinant bacteria carrying certain restriction fragments bordering the cmt operon were found to transform indole to indigo. This reaction, known to be catalyzed by toluene 2,3-dioxygenase, led to the discovery that the tod operon, encoding the catabolism of toluene, is located 2.8 kb downstream from and in the same orientation as the cmt operon in P. putida F1.
Regulators of aerobic and anaerobic respiration in Bacillus subtilis
Authors: Sun G , Sharkova E , Chesnut R , Birkey S , Duggan M F , Sorokin A , Pujic P , Ehrlich S D , Hulett F M
Published: November 2016

Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.
Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis
Authors: Mechold U , Cashel M , Steiner K , Gentry D , Malke H
Published: November 2016

We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.
Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa
Authors: Yahr T L , Barbieri J T , Frank D W
Published: November 2016

Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein. To determine the genetic relationship between the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by allelic exchange. A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS. Thirteen clones that cross-hybridized with the exoS probe were identified. One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS. Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of exoenzyme S are encoded by separate genes. In addition, this is the first report of the factor-activating-exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of exoenzyme S.
Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein
Authors: Johnson E P , Strom A R , Helinski D R
Published: November 2016

The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear.
The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain
Authors: Markham P N , Ahmed M , Neyfakh A A
Published: November 2016

Rhodamine and tetraphenylphosphonium, the substrates of the Bacillus subtilis multidrug efflux transporter Bmr, induce the expression of Bmr through direct interaction with its transcriptional activator BmrR. Here we show that the C-terminal domain of BmrR, expressed individually, binds both these compounds and therefore can be used as a model for molecular analysis of the phenomenon of multidrug recognition.
Periplasmic copper-zinc superoxide dismutase of Legionella pneumophila: role in stationary-phase survival
Authors: St John G , Steinman H M
Published: November 2016

Copper-zinc superoxide dismutases (CuZnSODs) are infrequently found in bacteria although widespread in eukaryotes. Legionella pneumophila, the causative organism of Legionnaires' disease, is one of a small number of bacterial species that contain a CuZnSOD, residing in the periplasm, in addition to an iron SOD (FeSOD) in their cytoplasm. To investigate CuZnSOD function, we purified the enzyme from wild-type L. pneumophila, obtained amino acid sequence data from isolated peptides, cloned and sequenced the gene from a L. pneumophila library, and then constructed and characterized a CuZnSOD null mutant. In contrast to the cytoplasmic FeSOD, the CuZnSOD of L. pneumophila is not essential for viability. However, CuZnSOD is critical for survival during the stationary phase of growth. The CuZnSOD null mutant survived 10(4)- to 10(6)-fold less than wild-type L. pneumophila. In wild-type L. pneumophila, the specific activity of CuZnSOD increased during the transition from exponential to stationary-phase growth while the FeSOD activity was constant. These data support a role of periplasmic CuZnSOD in survival of L. pneumophila during stationary phase. Since L. pneumophila survives extensive periods of dormancy between growth within hosts. CuZnSOD may contribute to the ability of this bacterium to be a pathogen. In exponential phase, wild-type and CuZnSOD null strains grew with comparable doubling times. In cultured HL-60 and THP-1 macrophage-like cell lines and in primary cultures of human monocytes, multiplication of the CuZnSOD null mutant was comparable to that of wild type. This indicated that CuZnSOD is not essential for intracellular growth within macrophages or for killing of macrophages in those systems.
Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli
Authors: Muffler A , Traulsen D D , Lange R , Hengge-Aronis R
Published: November 2016

The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth. Osmotic induction of rpoS can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine. Stimulation of rpoS translation and a change in the half-life of sigma(s) from 3 to 50 min both contribute to osmotic induction. Experiments with lacZ fusions inserted at different positions within the rpoS gene indicate that an element required for sigma(s) degradation is encoded between nucleotides 379 and 742 of the rpoS coding sequence.
Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis
Authors: Virlogeux I , Waxin H , Ecobichon C , Lee J O , Popoff M Y
Published: November 2016

Synthesis of Vi antigen, a capsular polysaccharide expressed by Salmonella typhi, is controlled by the viaA and viaB chromosomal loci. It was previously shown that Vi antigen expression was regulated by a system similar to the rcs regulatory system involved in colanic acid synthesis in Escherichia coli. We have cloned the rcsA, rcsB, and rcsC genes from S. typhi. The predicted amino sequences of the RcsA and RcsB proteins showed a high degree of similarity to their E. coli homologs. The nucleotide sequence of the rcsC gene was partially determined and was shown to be homologous to that of its E. coli counterpart. Complementation experiments indicated that rcsB and rcsC were encompassed within the viaA locus. The RcsA protein was not involved in Vi antigen synthesis. In contrast, the RcsB protein acted as a positive regulator of Vi polysaccharide expression. By mRNA and gene fusion analyses, we studied the role of RcsB and TviA, a via-B-encoded regulatory protein characterized previously, in regulating Vi antigen synthesis. The transcriptional start point of tviA mRNA was not influenced by RcsB or TviA. In the absence of RcsB or TviA protein, transcription of tviA gave rise to only a monocistronic tviA-specific mRNA. The presence of RcsB and TriA not only increased the amount of monocistronic tviA-specific mRNA but also resulted in countranscription of tviA and tviB, which is located immediately downstream of tviA on the viaB locus. In addition, TviA protein did not appear to be subject to degradation by the Lon protease. These results strongly suggest that TviA might act in concert with RcsB at the tviA promoter to activate transcription of the genes involved in Vi polymer synthesis in S. typhi in a Lon-independent manner.