bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA
Authors: Ellefson D D , Weber U , Wolfe A J
Published: November 2016

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.
Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri
Authors: Ferguson D J , Krzycki J A
Published: November 2016

Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction.
Effect of lipopolysaccharide mutations on recipient ability of Salmonella typhimurium for incompatibility group H plasmids
Authors: Sherburne C , Taylor D E
Published: November 2016

Previous investigations of the incompatibility group F, P, and I plasmid systems revealed the important role of the outer membrane components in the conjugal transfer of these plasmids. We have observed variability in transfer frequency of three incompatibility group H plasmids (IncHI1 plasmid R27, IncHI2 plasmid R478, and a Tn7 derivative of R27, pDT2454) upon transfer into various Salmonella typhimurium lipopolysaccharide (LPS) mutants derived from a common parental strain, SL1027. Recipients with truncated outer core via the rfaF LPS mutation increased the transfer frequency of the IncH plasmids by up to a factor of 10(3). Mutations which resulted in the truncation of the residues following 3-deoxy-D-manno-octulosonic acid, such as the rfaE and rfaD mutations, decreased the transfer frequency to undetectable levels. Addition of phosphorylethanolamine, a component of wild-type LPS, to the media decreased the frequency of transfer of R27 into wild-type and rfaF LPS mutant recipients tested. Reversing the direction of transfer, by mating LPS mutant donors with wild-type recipients, did not affect the frequency of transfer compared to the standard matings of wild-type donor with LPS mutant recipient. These findings demonstrate that conjugation interactions affected by LPS mutation are not specific for the recipient cell. Our results suggest that LPS mutation does not affect conjugation via altered pilus binding but affects some later steps in the conjugative process, and alteration of transfer frequency by O-phosphorylethanolamine and LPS truncation is due to charge-related interactions between the donor and recipient cell.
Antigenic variation in Neisseria gonorrhoeae: production of multiple lipooligosaccharides
Authors: Burch C L , Danaher R J , Stein D C
Published: November 2016

Individual cells of Neisseria gonorrhoeae may express a single lipooligosaccharide (LOS) component on their cell surfaces, or they may simultaneously express multiple LOS structures. Strain FA19 expresses LOS components that react with monoclonal antibodies (MAbs) 2-1-L8 and 1B2. The genetic locus responsible for this phenotype in FA19 was identified by isolating a clone that is able to impart the ability to simultaneously express both LOS molecules to strain 1291, a strain expressing only the MAb 1B2-reactive LOS. This clone, pCLB1, was characterized, and the gene responsible for the expression of both LOS components was determined to be lsi2. DNA sequence analysis of lsi2(Fa19) indicates that there are several differences between the DNA sequences of lsi2(FA19) and lsi2(1291). The region responsible for the LOS-specific phenotype change in lsi2(FA19) was identified by deletion and transformation analysis, mapping to a polyguanine tract within lsi2 where lsi2(FA19) possesses a +2 frameshift relative to lsi2(1291). The polyguanine tract in lsi2(FA19) was modified by site-directed mutagenesis to change the sequence to GGGAGGTGGCGGA to prevent frameshifting during DNA replication, transcription, and/or translation. Transformants of strain 1291 containing this DNA sequence express a single MAb 2-1-L8-reactive LOS component, the same phenotype exhibited by lsi2-defective strains. These data indicate that FA19 is able to generate a small amount of functional Lsi2 protein via transcriptional and/or translational frameshifting, and this limited amount of protein allows for the expression of MAb 1B2-reactive LOS molecules.
Mutational analysis of protein binding sites involved in formation of the bacteriophage lambda attL complex
Authors: MacWilliams M , Gumport R I , Gardner J F
Published: November 2016

Bacteriophage lambda site-specific recombination requires the formation of higher-order protein-DNA complexes to accomplish synapsis of the partner attachment (att) sites as well as for the regulation of the integration and excision reactions. The att sites are composed of a core region, the actual site of strand exchange, and flanking arm regions. The attL site consists of two core sites (C and C'), an integration host factor (IHF) binding site (H'), and three contiguous Int binding arm sites (P'1, P'2, and P'3). In this study, we employed bacteriophage P22 challenge phages to determine which protein binding sites participate in attL complex formation in vivo. The C', H', and P'1 sites were critical, because mutations in these sites severely disrupted formation of the attL complex. Mutations in the C and P'2 sites were less severe, and alteration of the P'3 site had no effect on complex formation. These results support a model in which IHF, bound to the H' site, bends the attL DNA so that the Int molecule bound to P'1 also interacts with the C' core site. This bridged complex, along with a second Int molecule bound to P'2, helps to stabilize the interaction of a third Int with the C core site. The results also indicate that nonspecific DNA binding is a significant component of the Int-core interactions and that the cooperativity of Int binding can overcome the effects of mutations in the individual arm sites and core sites.
Saccharomyces cerevisiae exhibits a yAP-1-mediated adaptive response to malondialdehyde
Authors: Turton H E , Dawes I W , Grant C M
Published: November 2016

Malondialdehyde (MDA) is a highly reactive aldehyde generally formed as a consequence of lipid peroxidation. MDA has been inferred to have mutagenic and cytotoxic roles and possibly to be a participant in the onset of atherosclerosis. Wild-type Saccharomyces cerevisiae acquires resistance to a lethal dose (5 mM) of MDA following prior exposure to a nonlethal concentration (1 mM). This response was completely inhibited by cycloheximide (50 microg ml(-1)), indicating a requirement for protein synthesis for adaptation. Furthermore, we have examined the roles of glutathione (GSH), mitochondrial function, and yAP-1-mediated transcription in conferring resistance and adaptation to MDA. A yap1 disruption mutant exhibited the greatest sensitivity and was unable to adapt to MDA, implicating yAP-1 in both the adaptive response and constitutive survival. The effect of MDA on GSH mutants indicated a role for GSH in initial resistance, whereas resistance acquired through adaptation was independent of GSH. Likewise, respiratory mutants (petite mutants) were sensitive to MDA but were still able to mount an adaptive response similar to that of the wild type, excluding mitochondria from any role in adaptation. MDA was detected in yeast cells by the thiobarbituric acid test and subsequent high-pressure liquid chromatography separation. Elevated levels were detected following treatment with hydrogen peroxide. However, the MDA-adaptive response was independent of that to H2O2.
Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans
Authors: Tsukioka Y , Yamashita Y , Oho T , Nakano Y , Koga T
Published: November 2016

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria. The cell extract of Escherichia coli expressing each of the cloned genes of S. mutans exhibited enzymatic activity corresponding to the homologous counterpart of the rfb gene products. Rhamnose was not detected in the cell wall preparation purified from the mutant in which each of the three cloned genes was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with the autoclaved extracts from these mutants. These results indicate that the gene products identified in the present study are involved in the dTDP-L-rhamnose synthesis pathway and that the pathway relates to the biosynthesis of the serotype-specific polysaccharide antigen of S. mutans. Southern hybridization analysis revealed that genes homologous to the cloned genes involved in the dTDP-L-rhamnose synthesis pathway were widely distributed in a variety of streptococci. This is the first report of the biological function of the dTDP-rhamnose pathway in streptococci.
The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens
Authors: Baron C , Thorstenson Y R , Zambryski P C
Published: November 2016

VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants. In this report, we present a biochemical analysis of their interaction and cellular localization. A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins. Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers. Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex. Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues. The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system. Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with [3H]palmitic acid in A. tumefaciens verified that VirB7 is a lipoprotein associated with VirB9. VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex.
Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis
Authors: Kutsukake K
Published: November 2016

During flagellar morphogenesis in Salmonella typhimurium, the genes involved in filament assembly are expressed fully only after completion of hook-basal body assembly. This coupling of gene expression to morphogenesis is achieved by exporting the flagellum-specific anti-sigma factor, FlgM, out of the cell through the mature hook-basal body structure. Therefore, the flagellum-specific export apparatus must be able to sense the assembly state of the flagellar structure and to turn on FlgM export at a specific stage of hook assembly. It has been suggested that FlhB may act as the molecular switch which mediates this ordered export. Here, I report genetic evidence that in addition to FlhB, the product of a newly identified gene, rflH, is involved in the negative regulation of FlgM export. FlgM is released through the basal body structure lacking the hook and the filament only when the flhB and rflH genes are both defective. Therefore, the export gate for FlgM should be double locked by FlhB and RflH. The rflH gene is located at around 52 min, where no flagellum-related gene has been found. I propose a revised model of the export-switching machinery which consists of two systems, the hook-length signal transduction pathway and the double-locked gate for FlgM export.
Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca2+ response
Authors: Nilles M L , Williams A W , Skrzypek E , Straley S C
Published: November 2016

Yersinia pestis contains a virulence plasmid, pCD1, that encodes many virulence-associated traits, such as the Yops (Yersinia outer proteins) and the bifunctional LcrV, which has both regulatory and antihost functions. In addition to LcrV and the Yops, pCD1 encodes a type III secretion system that is responsible for Yop and LcrV secretion. The Yop-LcrV secretion mechanism is believed to regulate transcription of lcrV and yop operons indirectly by controlling the intracellular concentration of a secreted repressor. The activity of the secretion mechanism and consequently the expression of LcrV and Yops are negatively regulated in response to environmental conditions such as Ca2+ concentration by LcrE and, additionally, by LcrG, both of which have been proposed to block the secretion mechanism. This block is removed by the absence of Ca2+ or by contact with eukaryotic cells, and some Yops are then translocated into the cells. Regulation of LcrV and Yop expression also is positively affected by LcrV. Previously, LcrG was shown to be secreted from bacterial cells when the growth medium lacks added Ca2+, although most of the LcrG remains cell associated. In the present study, we showed that the cell-associated LcrG is cytoplasmically localized. We demonstrated that LcrG interacts with LcrV to form a heterodimeric complex by using chemical cross-linking and copurification of LcrG and LcrV. Additionally, we found that small amounts of LcrV and YopE can be detected in periplasmic fractions isolated by cold osmotic shock and spheroplast formation, indicating that their secretion pathway is accessible to the periplasm or to these procedures for obtaining periplasmic fractions. We propose that the cytoplasmically localized LcrG blocks the Yop secretion apparatus from the cytoplasmic side and that LcrV is required to remove the LcrG secretion block to yield full induction of Yop and LcrV secretion and expression.
Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen
Authors: Bereswill S , Geider K
Published: November 2016

RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.
Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis
Authors: Skinner L M , Jackson M P
Published: November 2016

The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.
Evidence that KpsT, the ATP-binding component of an ATP-binding cassette transporter, is exposed to the periplasm and associates with polymer during translocation of the polysialic acid capsule of Escherichia coli K1
Authors: Bliss J M , Silver R P
Published: November 2016

KpsT utilizes ATP to effect translocation of the polysialic acid capsule of Escherichia coli K1. We have previously proposed a mechanistic model for the action of this protein. Here, we provide evidence to support two predictions of the model: that KpsT associates with polymer and that KpsT is accessible from the periplasmic surface of the inner membrane.
Role of His243 in the phosphatase activity of EnvZ in Escherichia coli
Authors: Skarphol K , Waukau J , Forst S A
Published: November 2016

EnvZ undergoes autophosphorylation at His243 and subsequently transfers the phosphate group to OmpR. EnvZ also possesses an OmpR-phosphate phosphatase activity. We examined the role of His243 in the phosphatase function by replacing His with either Val, Tyr, Ser, Asp, or Asn. EnvZH243V and EnvZH243Y were both shown to possess phosphatase activity in vitro. In addition, the mutant proteins were able to reduce the high level of OmpR-phosphate present in the envZ473 strain. These results indicate that His243 of EnvZ is not essential for stimulating the dephosphorylation of OmpR-phosphate.
bmr3, a third multidrug transporter gene of Bacillus subtilis
Authors: Ohki R , Murata M
Published: November 2016

A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase.
An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels
Authors: Hassett D J , Howell M L , Ochsner U A , Vasil M L , Johnson Z , Dean G E
Published: November 2016

The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.
Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus
Authors: Yakunin A F , Hallenbeck P C
Published: November 2016

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.
Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723
Authors: Lee J Y , Xun L
Published: November 2016

The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.
Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole
Authors: Horak J , Wolf D H
Published: November 2016

When Saccharomyces cerevisiae cells growing on galactose are transferred onto glucose medium containing cycloheximide, an inhibitor of protein synthesis, a rapid reduction of Gal2p-mediated galactose uptake is observed. We show that glucose-induced inactivation of Gal2p is due to its degradation. Stabilization of Gal2p in pra1 mutant cells devoid of vacuolar proteinase activity is observed. Subcellular fractionation and indirect immunofluorescence showed that the Gal2 transporter accumulates in the vacuole of the mutant cells, directly demonstrating that its degradation requires vacuolar proteolysis. In contrast, Gal2p degradation is proteasome independent since its half-life is unaffected in pre1-1 pre2-2, cim3-1, and cim5-1 mutants defective in several subunits of the protease complex. In addition, vacuolar delivery of Gal2p was shown to be blocked in conditional end3 and end4 mutants at the nonpermissive temperature, indicating that delivery of Gal2p to the vacuole occurs via the endocytic pathway. Taken together, the results presented here demonstrate that glucose-induced proteolysis of Gal2p is dependent on endocytosis and vacuolar proteolysis and is independent of the functional proteasome. Moreover, we show that Gal2p is ubiquitinated under conditions of glucose-induced inactivation.
Pesticin displays muramidase activity
Authors: Vollmer W , Pilsl H , Hantke K , Höltje J V , Braun V
Published: November 2016

Pesticin of Yersinia pestis is the only bacteriocin that converts sensitive cells to stable spheroplasts. The amino acid sequence of pesticin as derived from the nucleotide sequence shows no similarity to those of any of the bacteriocins. The unique properties of pesticin prompted an investigation of its mode of action. Since the pesticin plasmid does not encode a lysis protein for release of pesticin into the culture medium, pesticin was isolated from cells and purified to electrophoretic homogeneity. Highly purified pesticin degraded murein and murein glycan strands lacking the peptide side chains to products that were similar to those obtained by lysozyme, as revealed by high-resolution high-pressure liquid chromatography. After reduction of the murein degradation products with tritium-labeled sodium borohydride, acid hydrolysis, and separation of the products by thin-layer chromatography, radiolabeled muraminitol was identified. This indicates that pesticin is a muramidase, and not an N-acetyl-glucosaminidase, that converts cells into stable spheroplasts by slowly degrading murein.
Molecular characterization and transcriptional analysis of type 8 capsule genes in Staphylococcus aureus
Authors: Sau S , Sun J , Lee C Y
Published: November 2016

A 20.5-kb contiguous DNA fragment from Staphylococcus aureus Becker affecting type 8 capsule (CP8) biosynthesis was previously cloned. Sequencing analysis indicated that 16 open reading frames (ORFs) encoded within this fragment might be involved in CP8 synthesis. Using various plasmids containing DNA inserts derived from the 20.5-kb region, we showed by complementation of chemical mutants that 8 of the 16 ORFs were required for CP8 synthesis. To determine the involvement of the remaining eight ORFs, nonpolar gene-specific chromosomal mutations located in each of these ORFs were constructed. We found that three additional ORFs were also involved in the CP8 synthesis. Thus, 11 of the 16 ORFs were shown to affect CP8 synthesis. Complementation analyses of these 11 type 8 capsule (cap8) genes affecting CP8 production showed several promoters within the cap8 gene cluster. However, by Northern hybridization using either the entire cap8 gene cluster or the internal fragments of individual ORFs as probes, one 17-kb cap8-specific transcript was detected. Using xylE as the reporter gene, we found that the promoter at the beginning of the cap8 operon was much stronger than any of the internal promoters. These results suggest that the cap8 genes are transcribed mainly as a single large transcript. In addition, Southern hybridization analyses showed that cap8H, cap8I, cap8J, and cap8K, located in the central region of the cap8 gene cluster, were CP8 specific.
Functional analysis of exsC and exsB in regulation of exoenzyme S production by Pseudomonas aeruginosa
Authors: Goranson J , Hovey A K , Frank D W
Published: November 2016

Expression of ExsC, ExsB, and ExsA (the exoenzyme S trans-regulatory locus) of Pseudomonas aeruginosa was analyzed by using complementation, RNase protection, translational fusion, and T7-directed protein expression analyses. T7 expression analyses in E. coli hosts demonstrated that ExsC, ExsA, and a truncated form of ExsD (a partial open reading frame located 3' of ExsA) were translated; however, a product corresponding to ExsB was undetectable. T7-mediated transcription and translation of the antisense strand resulted in production of a 18.5-kDa product, termed ExsB', which overlapped the predicted ExsB product. In complementation experiments, deletion of the region encoding ExsB and most of ExsB' severely reduced exoenzyme S production. Site-specific mutagenesis of the start codons for ExsB and ExsB', however, did not affect exoenzyme S production. RNase protection studies were initiated to examine the hypothesis that RNA encoded within the ExsB/ExsB' region exerted a regulatory effect. RNA encoding ExsB' was not detectable from chromosomal genes or complementation constructs, indicating that ExsB' was not expressed in P. aeruginosa. To determine the pattern of translation, a chloramphenicol acetyltransferase gene (cat) reporter was fused in frame with ExsB and with ExsA in the context of the entire locus or in the absence of the exsB region. These experiments indicated that exsB was not translated but that deletion of the exsB region affected the translation of ExsA-CAT. RNase protection assays further suggested that deletion of exsB resulted in a processing of ExsA mRNA. Our data indicate that the untranslated exsB region of the trans-regulatory locus mRNA mediates either the stability or the translation of exsA. Complementation analysis further suggests that ExsC may play a role in the translation or stability of ExoS.
A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species
Authors: Lenz O , Strack A , Tran-Betcke A , Friedrich B
Published: November 2016

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2. This repression was released by truncation of hoxJ. H2-dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.
The divIVA minicell locus of Bacillus subtilis
Authors: Cha J H , Stewart G C
Published: November 2016

The Bacillus subtilis divIVA1 mutation causes misplacement of the septum during cell division, resulting in the formation of small, circular, anucleate minicells. This study reports the cloning and sequence analysis of 2.4 kb of the B. subtilis chromosome including the divIVA locus. Three open reading frames were identified: orf, whose function is unknown; divIVA; and isoleucyl tRNA synthetase (ileS). We identified the point mutation in the divIVA1 mutant allele. Inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype. Mutants with mutations at both of the minicell loci of B. subtilis, divIVA and divIVB, possess a minicell phenotype identical to that of the DivIVB- mutant. The DivIVA-mutants, but not the DivIVB- mutants, show a decrease in sporulation efficiency and a delay in the kinetics of endospore formation. The data support a model in which divIVA encodes the topological specificity subunit of the minCD system. The model suggests that DivIVA acts as a pilot protein, directing minCD to the polar septation sites. DivIVA also appears to be the interface between a sporulation component and MinCD, freeing up the polar septation sites for use during the asymmetric septation event of the sporulation process.
Characterization of DinR, the Bacillus subtilis SOS repressor
Authors: Winterling K W , Levine A S , Yasbin R E , Woodgate R
Published: November 2016

In Bacillus subtilis, exposure to DNA damage and the development of natural competence lead to the induction of the SOS regulon. It has been hypothesized that the DinR protein is the cellular repressor of the B. subtilis SOS system due to its homology to the Escherichia coli LexA transcriptional repressor. Indeed, comparison of DinR and its homologs from gram-negative and -positive bacteria revealed conserved structural motifs within the carboxyl-terminal domain that are believed to be important for autocatalysis of the protein. In contrast, regions within the DNA binding domain were conserved only within gram-negative or -positive genera, which possibly explains the differences in the sequence specificities between gram-negative and gram-positive SOS boxes. The hypothesis that DinR is the repressor of the SOS regulon in B. subtilis has been tested through overexpression, purification, and characterization of the DinR protein. Like E. coli LexA, B. subtilis DinR undergoes an autocatalytic reaction at alkaline pH at a siscile Ala91-Gly92 bond. The cleavage reaction can also be mediated in vitro under more physiological conditions by the E. coli RecA protein. By using electrophoretic mobility shift assays, we demonstrated that DinR interacts with the previously characterized SOS box of the B. subtilis recA gene, but not with sequences containing single base pair mutations within the SOS box. Together, these observations strongly suggest that DinR is the repressor of the SOS regulon in B. subtilis.
Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum
Authors: Das A , Ivey D M , Ljungdahl L G
Published: November 2016

The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium. The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E. coli.
The major outer membrane protein of Haemophilus ducreyi consists of two OmpA homologs
Authors: Klesney-Tait J , Hiltke T J , Maciver I , Spinola S M , Radolf J D , Hansen E J
Published: November 2016

The major outer membrane protein (MOMP) of Haemophilus ducreyi is an OmpA homolog that migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as three species with apparent molecular weights ranging from 37,000 to 43,000. Monoclonal antibodies directed against this macromolecule were used to identify recombinant clones containing fragments of the gene encoding this protein. Nucleotide sequence analysis of these fragments confirmed that the MOMP encoded by the intact gene (momp) was a member of the OmpA family of outer membrane proteins. Construction of an isogenic H. ducreyi mutant unable to express the MOMP led to the discovery of a second outer membrane protein which migrated at the same rate on SDS-PAGE gels as the MOMP. N-terminal amino acid sequence analysis of this second protein revealed that its N terminus was nearly identical to that of the MOMP and also had homology with members of the OmpA family. Nucleotide sequence analysis of the region downstream from the momp gene revealed the presence of a partial open reading frame encoding a predicted OmpA-like protein. A modification of anchored PCR technology was used to obtain the nucleotide sequence of this downstream gene which was shown to encode a second OmpA homolog (OmpA2). The N-terminal amino acid sequence of OmpA2 was identical to that of the OmpA-like protein detected in the momp mutant. The H. ducreyi MOMP and OmpA2 proteins, which comigrated on SDS-PAGE gels and which were encoded by the tandem arranged momp and ompA2 genes, were 72% identical.
The Salmonella typhimurium mar locus: molecular and genetic analyses and assessment of its role in virulence
Authors: Sulavik M C , Dazer M , Miller P F
Published: November 2016

The marRAB operon is a regulatory locus that controls multiple drug resistance in Escherichia coli. marA encodes a positive regulator of the antibiotic resistance response, acting by altering the expression of unlinked genes. marR encodes a repressor of marRAB transcription and controls the production of MarA in response to environmental signals. A molecular and genetic study of the homologous operon in Salmonella typhimurium was undertaken, and the role of marA in virulence in a murine model was assessed. Expression of E. coli marA (marAEC) present on a multicopy plasmid in S. typhimurium resulted in a multiple antibiotic resistance (Mar) phenotype, suggesting that a similar regulon exists in this organism. A genomic plasmid library containing S. typhimurium chromosomal sequences was introduced into an E. coli strain that was deleted for the mar locus and contained a single-copy marR'-'lacZ translational fusion. Plasmid clones that contained both S. typhimurium marR (marRSt) and marA (marASt) genes were identified as those that were capable of repressing expression of the fusion and which resulted in a Mar phenotype. The predicted amino acid sequences of MarRSt, MarASt, and MarBSt were 91, 86, and 42% identical, respectively, to the same genes from E. coli, while the operator/promoter region of the operon was 86% identical to the same 98-nucleotide-upstream region in E. coli. The marRAB transcriptional start sites for both organisms were determined by primer extension, and a marRABSt transcript of approximately 1.1 kb was identified by Northern blot analysis. Its accumulation was shown to be inducible by sodium salicylate. Open reading frames flanking the marRAB operon were also conserved. An S. typhimurium marA disruption strain was constructed by an allelic exchange method and compared to the wild-type strain for virulence in a murine BALB/c infection model. No effect on virulence was noted. The endogenous S. typhimurium plasmid that is associated with virulence played no role in marA-mediated multiple antibiotic resistance. Taken together, the data show that the S. typhimurium mar locus is structurally and functionally similar to marRABEc and that a lesion in marASt has no effect on S. typhimurium virulence for BALB/c mice.
Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1
Authors: Pontarollo R A , Rioux C R , Potter A A
Published: November 2016

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study.