bluuebunny/crossref_metadata_2025 huggingface dataset:
cobB function is required for catabolism of propionate in Salmonella typhimurium LT2: evidence for existence of a substitute function for CobB within the 1,2-propanediol utilization (pdu) operon
Authors: Tsang A W , Escalante-Semerena J C
Published: November 2016

The cobB function of Salmonella typhimurium LT2 was defined in vivo as an alternative activity for the nicotinic acid mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme (CobT), which is involved in the assembly of the nucleotide loop of cobalamin in this bacterium (J. R. Trzebiatowski, G. A. O'Toole, and J. C. Escalante-Semerena, J. Bacteriol. 176:3568-3575, 1994). In this paper we document that, independent of their inability to substitute for CobT function, cobB mutants are unable to use propionate as a carbon and energy source. A plasmid carrying only a wild-type copy of cobB complemented the cobalamin biosynthesis and propionate catabolism phenotypes of cobB mutants, indicating that a lack of CobB was responsible for both phenotypes. We demonstrate the existence of a function encoded by the 1,2-propanediol utilization (pdu) operon, which when induced by 1,2-propanediol compensated for the lack of CobB during propionate catabolism but failed to compensate for CobT in the assembly of the nucleotide loop of cobalamin in a cobB cobT double mutant.
The linker region of AraC protein
Authors: Eustance R J , Schleif R F
Published: November 2016

AraC protein, a transcriptional regulator of the L-arabinose operon in Escherichia coli, is dimeric. Each monomer consists of a domain for DNA binding plus transcription activation and a domain for dimerization plus arabinose binding. These are connected to one another by a linker region of at least 5 amino acids. Here we have addressed the question of whether any of the amino acids in the linker region play active, specific, and crucial structural roles or whether these amino acids merely serve as passive spacers between the functional domains. We found that all but one of the linker amino acids can be changed to other amino acids individually and in small groups without substantially affecting the ability of AraC protein to activate transcription when arabinose is present. When, however, the entire linker region is replaced with linker sequences from other proteins, the functioning of AraC is impaired.
Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium
Authors: Toker A S , Kihara M , Macnab R M
Published: November 2016

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.
The levanase operon of Bacillus subtilis expressed in Escherichia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection
Authors: Martin-Verstraete I , Michel V , Charbit A
Published: November 2016

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane. LamB also serves as a receptor for several other bacteriophages. Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E. coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS transporters for mannose of E. coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters. These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins. In the present work, we show that the fructose-specific permease encoded by the levanase operon of B. subtilis is inducible by mannose and allows mannose uptake in B. subtilis as well as in E. coli. Moreover, we show that the B. subtilis permease can substitute for the E. coli mannose permease cytoplasmic membrane components for phage lambda infection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.
Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis
Authors: Sundin G W , Shankar S , Chakrabarty A M
Published: November 2016

We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as critical to this process.
Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa)
Authors: Gage D J , Bobo T , Long S R
Published: November 2016

A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R. meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads. In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light. GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R. meliloti residing inside its host plant.
Regulation of pelZ, a gene of the pelB-pelC cluster encoding a new pectate lyase of Erwinia chrysanthemi 3937
Authors: Pissavin C , Robert-Baudouy J , Hugouvieux-Cotte-Pattat N
Published: November 2016

The phytopathogenic enterobacterium Erwinia chrysanthemi 3937 produces five major and several secondary endo-pectate lyases encoded by the pel genes. Most of these genes are arranged in clusters on the bacterial chromosome. The genomic region surrounding the pelB-pelC cluster was supposed to be involved in the regulation of PelB and PelC synthesis. We demonstrated that the variation of pelB expression resulted from the titration of a regulatory protein by the gene adjacent to pelC. This gene was renamed pelZ since it encodes a protein of 420 amino acids with an endo-pectate lyase activity. Regulation of pelZ expression was investigated by using transcriptional fusions and a study of mRNA synthesis. Its transcription depends on different environmental conditions. It is induced in planta and in the presence of pectic catabolite products. This induction seems to be partially mediated by the KdgR protein but does not result from a direct interaction of KdgR with the pelZ 5' region. The transcription of pelZ leads to the synthesis of a monocistronic mRNA. However, the synthesis of a polycistronic mRNA from the pelC promoter, regulated by KdgR, is responsible for increased production of PelZ under inducing conditions. pelZ transcription is also controlled by pecT, which regulates some other pel genes, but it is independent of the pecS regulatory locus. The pelZ gene appears to be widespread in different strains of E. chrysanthemi. Moreover, a gene homologous to pelZ exists in Erwinia carotovora subsp. atroseptica adjacent to the cluster containing the pectate lyase-encoding genes pel1, pel2, and pel3. This conservation could reflect a significant role of PelZ in the pectinolytic system of Erwiniae. We showed pelZ is not a predominant virulence factor of E. chrysanthemi but is involved in host specificity.
Lrp is a direct repressor of the dad operon in Escherichia coli
Authors: Mathew E , Zhi J , Freundlich M
Published: November 2016

Expression of the degradative D-amino acid dehydrogenase (dad) operon is known to be increased when Escherichia coli is grown in the presence of D- or L-alanine. Alanine is thought to act as an inducer to block the action of a postulated repressor. This operon is also believed to be regulated by catabolite repression. We have used in vivo and in vitro experiments that show that the dad repressor is the leucine-responsive regulatory protein (Lrp). dad expression in a dad-lacZ operon fusion strain was increased four- to sevenfold when cells were grown in minimal medium containing alanine or leucine. A strain lacking Lrp had high-level constitutive dad expression. Gel retardation and footprinting studies revealed that Lrp binds in vitro to multiple sites over a large area in the dad promoter region. This binding was reduced by alanine or leucine. In vitro transcription assays, using a plasmid template and primer extension analysis, identified three major dad transcripts (Tr1, Tr2, and Tr3). The formation of these transcripts was differentially regulated by cyclic AMP-cyclic AMP receptor protein complex, and each was strongly repressed by Lrp. Alanine or leucine completely (for Tr1 and Tr2) or partially (for Tr3) reversed Lrp inhibition. Site-directed mutagenesis of an Lrp binding site strongly reduced Lrp binding and prevented Lrp repression of dad transcription in vivo and in vitro. Taken together, these results strongly suggest that Lrp and alanine or leucine act directly to repress and induce, respectively, transcription of the dad operon.
Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2)
Authors: Shima J , Hesketh A , Okamoto S , Kawamoto S , Ochi K
Published: November 2016

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.
Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii
Authors: Premakumar R , Jacobitz S , Ricke S C , Bishop P E
Published: November 2016

A tungsten-tolerant mutant strain (CA6) of Azotobacter vinelandii first described in 1980 (P. E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980) has been further characterized. Results from growth experiments suggest that both nitrogenases 1 and 3 are utilized when CA6 grows in N-free medium containing Na2MoO4. Strain CA6.1.71, which lacks both nitrogenases 2 and 3, grew as well as strain CA in N-free medium containing Na2MoO4 after an initial lag. This indicates that nitrogenase 1 is fully functional in strain CA6. nifH-lacZ and anfH-lacZ transcriptional fusions were expressed in CA6 in the presence of Na2MoO4. Thus, in contrast to wild-type strain CA, transcription of the anfHDGK gene cluster in strain CA6 is not repressed by Mo. Expression of the vnfD-lacZ fusion was the same in both strains CA and CA6. In agreement with the results obtained with lac fusions, subunits of both nitrogenases 1 and 3 were found in protein extracts of CA6 cells grown in N-free medium containing Na2MoO4. However, CA6 cells, cultured in the presence of Na2WO4, accumulated nitrogenase 3 proteins without detectable amounts of nitrogenase 1 proteins. This indicates that expression of Mo-independent nitrogenase 3 is the basis for the tungsten tolerance phenotype of strain CA6. A measure of Mo accumulation as a function of time showed that accumulation by strain CA6 was slower than that for strain CA. When Mo accumulation was studied as a function of Na2MoO4 concentration, the two strains accumulated similar amounts of Mo in the concentration range of 0 to 1 microM Na2MoO4 during a 2-h period. Within the range of 1 to 5 microM Na2MoO4, Mo accumulation by strain CA increased linearly with increasing concentration whereas no further increases were observed for strain CA6. These results are consistent with the possibility that the tungsten tolerance mutation carried by CA6 is in a Mo transport system.
Transcriptional regulation of the Bacillus subtilis menp1 promoter
Authors: Qin X , Taber H W
Published: November 2016

The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone. The menp1 promoter previously was shown to be the primary cis element for menFD gene expression. In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase. The effect on menp1 activity by a particular end product (such as acetoin or acetate) was prevented by blocking the corresponding pathway for end product utilization. Alteration of a TGAAA motif within the promoter region resulted in unregulated menp1 activity throughout the culture cycle, irrespective of the carbon source added.
Low-affinity potassium uptake system in the archaeon Methanobacterium thermoautotrophicum: overproduction of a 31-kilodalton membrane protein during growth on low-potassium medium
Authors: Glasemacher J , Siebers A , Altendorf K , Schönheit P
Published: November 2016

During growth on low-K+ medium (1 mM K+), Methanobacterium thermoautotrophicum accumulated K+ up to concentration gradients ([K+]intracellular/[K+]extracellular) of 25,000- to 50,000-fold. At these gradients ([K+]extracellular of < 20 microM), growth ceased but could be reinitiated by the addition of K+ or Rb+. During K+ starvation, the levels of a protein with an apparent molecular weight of 31,000 increased about sixfold. The protein was associated with the membrane and could be extracted by detergents. Cell suspensions of M. thermoautotrophicum obtained after K+-limited growth catalyzed the transport of both K+ and Rb+ with apparent Km and Vmax values of 0.13 mM and 140 nmol/min/mg, respectively, for K+ and 3.4 mM and 140 nmol/min/mg, respectively, for Rb+. Rb+ competitively inhibited K+ uptake with an inhibitor constant of about 10 mM. Membranes of K+-starved cells did not exhibit K+-stimulated ATPase activity. Immunoblotting with antisera against Escherichia coli Kdp-ATPase did not reveal any specific cross-reactivity against membrane proteins of K+-starved cells. Cells of M. thermoautotrophicum grown at a high potassium concentration (50 mM) catalyzed K+ and Rb+ transport at similar apparent Km values (0.13 mM for K+ and 3.3 mM for Rb+) but at significantly lower apparent Vmax values (about 60 nmol/min/mg for both K+ and Rb+) compared with K+-starved cells. From these data, it is concluded that the archaeon M. thermoautotrophicum contains a low-affinity K+ uptake system which is overproduced during growth on low-K+ medium.
Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii
Authors: Ferdows M S , Serwer P , Griess G A , Norris S J , Barbour A G
Published: November 2016

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.
Spiroplasma citri virus SpV1-derived cloning vector: deletion formation by illegitimate and homologous recombination in a spiroplasmal host strain which probably lacks a functional recA gene
Authors: Marais A , Bové J M , Renaudin J
Published: November 2016

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for expressing an epitope of the P1 adhesin protein from Mycoplasma pneumoniae in S. citri (A. Marais, J. M. Bové, S.F. Dallo, J. B. Baseman, and J. Renaudin, J. Bacteriol. 175:2783-2787, 1993). We have now studied the structural instability of the recombinant RF leading to loss of the DNA insert. Analyses of viral clones with deletions have shown that both illegitimate and homologous recombination were involved in deletion formation. For one such clone, deletion has occurred via a double crossing-over exchange between the circular free viral RF and SpV1 viral sequences present in the S. citri host chromosome. The homologous recombination process usually requires the RecA protein. However, characterization of the recA gene of the S. citri R8A2 host strain revealed that over two-thirds of the open reading frame of the recA gene was deleted from the C-terminal part, indicating that this particular strain is probably RecA deficient.
Primary structure and phylogeny of the Calvin cycle enzymes transketolase and fructosebisphosphate aldolase of Xanthobacter flavus
Authors: van den Bergh E R , Baker S C , Raggers R J , Terpstra P , Woudstra E C , Dijkhuizen L , Meijer W G
Published: November 2016

Xanthobacter flavus, a gram-negative facultatively autotrophic bacterium, employs the Calvin cycle for the fixation of carbon dioxide. Cells grown under autotrophic growth conditions possess an Fe(2+)-dependent fructosebisphosphate (FBP) aldolase (class II) in addition to a class I FBP aldolase. By nucleotide sequencing and heterologous expression in Escherichia coli, genes encoding transketolase (EC 2.2.1.1.; CbbT) and class II FBP aldolase (EC 4.1.2.13; CbbA) were identified. A partial open reading frame encoding a protein similar to pentose-5-phosphate 3-epimerase was identified downstream from cbbA. A phylogenetic tree of transketolase proteins displays a conventional branching order. However, the class II FBP aldolase protein from X. flavus is only distantly related to that of E. coli. The autotrophic FBP aldolase proteins from X. flavus, Alcaligenes eutrophus, and Rhodobacter sphaeroides form a tight cluster, with the proteins from gram-positive bacteria as the closest relatives.
Negative regulation by fliD, fliS, and fliT of the export of the flagellum-specific anti-sigma factor, FlgM, in Salmonella typhimurium
Authors: Yokoseki T , Iino T , Kutsukake K
Published: November 2016

The fliD operon of Salmonella typhimurium consists of three flagellar genes, fliD, fliS, and fliT, and is transcribed in this order. It has been shown that an fliD::Tn10 mutation causes an excess export of the flagellum-specific anti-sigma factor, FlgM, resulting in an overexpression of flagellar class 3 operons. In this study, using gene-disruption mutants in the individual genes in the fliD operon, we showed that mutations in any one of the genes in the operon enhanced both FlgM export and the expression of flagellar regulon. This indicates that all three genes in the operon are involved in the negative regulation of FlgM export.
Further genetic analysis of the activation function of the TyrR regulatory protein of Escherichia coli
Authors: Yang J , Camakaris H , Pittard A J
Published: November 2016

Previous reports (J. Cui and R. L. Somerville, J. Bacteriol. 175:1777-1784, 1993; J. Yang, H. Camakaris, and A. J. Pittard, J. Bacteriol. 175:6372-6375, 1993) have identified a number of amino acids in the N-terminal domain of the TyrR protein which are critical for activation of gene expression but which play no role in TyrR-mediated repression. These amino acids were clustered in a single region involving positions 2, 3, 5, 7, 9, 10, and 16. Using random and site-directed mutagenesis, we have identified an additional eight key amino acids whose substitution results in significant or total loss of activator function. All of these are located in the N-terminal domain of TyrR. Alanine scanning at these eight new positions and at five of the previously identified positions for which alanine substitutions had not been obtained has identified three amino acids whose side chains are critical for activation, namely, D-9, R-10, and D-103. Glycine at position 37 is also of critical importance. Alanine substitutions at four other positions (C-7, E-16, D-19, and V-93) caused partial but significant loss of activation, indicating that the side chains of these amino acids also play a contributing role in the activation process.
Suppression of ftsH mutant phenotypes by overproduction of molecular chaperones
Authors: Shirai Y , Akiyama Y , Ito K
Published: November 2016

Decreased intracellular levels of FtsH, a membrane-bound ATPase, led to retardation of growth and protein export, as well as to an abnormal translocation of alkaline phosphatase that had been attached to a cytoplasmic domain of a multispanning membrane protein, SecY. The last phenotype is designated Std (stop transfer defective). In this study, we examined the effects of overproduction of some molecular chaperones on the phenotypes of ftsH mutants. The growth retardation was partially suppressed by overproduction of GroEL/GroES (Hsp60/Hsp10) or HtpG (Hsp90), although these chaperones could not totally substitute for FtsH. Overproduction of HtpG specifically alleviated the Std phenotype, while that of GroEL/GroES alleviated the protein export defect of ftsH mutants. These results suggest that FtsH functions can be somehow compensated for when the cellular concentrations of some molecular chaperones increase.
CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in beta 1,6-glucan assembly
Authors: Jiang B , Sheraton J , Ram A F , Dijkgraaf G J , Klis F M , Bussey H
Published: November 2016

CWH41 encodes a novel type II integral membrane N-glycoprotein located in the endoplasmic reticulum. Disruption of the CWH41 gene leads to a K1 killer toxin-resistant phenotype and a 50% reduction in the cell wall beta 1,6-glucan level. CWH41 also displays strong genetic interactions with KRE1 and KRE6, two genes known to be involved in the beta 1,6-glucan biosynthetic pathway. The cwh41 delta kre6 delta double mutant is nonviable; and the cwh41 delta kre1 delta double mutation results in strong synergistic defects, with a severely slow-growth phenotype, a 75% reduction in beta 1,6-glucan level, and the secretion of a cell wall glucomannoprotein, Cwp1p. These results provide strong genetic evidence indicating that Cwh41p plays a functional role, possibly as a new synthetic component, in the assembly of cell wall beta 1,6-glucan.
Adherence, accumulation, and cell division of a natural adherent bacterial population
Authors: Bloomquist C G , Reilly B E , Liljemark W F
Published: November 2016

Developing dental bacterial plaques formed in vivo on enamel surfaces were examined in specimens from 18 adult volunteers during the first day of plaque formation. An intraoral model placing enamel pieces onto teeth was used to study bacterial plaque populations developing naturally to various cell densities per square millimeter of surface area of the enamel (W. F. Liljemark, C. G. Bloomquist, C. L. Bandt, B. L. Philstrom, J. E. Hinrichs, and L. F. Wolff, Oral Microbiol. Immunol. 8:5-15, 1993). Radiolabeled nucleoside incorporation was used to measure DNA synthesis concurrent with the taking of standard viable cell counts of the plaque samples. Results showed that in vivo plaque formation began with the rapid adherence of bacteria until ca. 12 to 32% of the enamel's salivary pellicle was saturated (ca. 2.5 x 10(5) to 6.3 x 10(5) cells per mm2). The pioneer adherent species were predominantly those of the "sanguis streptococci." At the above-noted density, the bacteria present on the salivary pellicle incorporated low levels of radiolabeled nucleoside per viable cell. As bacterial numbers reached densities between 8.0 x 10(5) and 2.0 x 10(6) cells per mm2, there was a small increase in the incorporation of radiolabeled nucleosides per cell. At 2.5 x 10(6) to 4.0 x 10(6) cells per mm2 of enamel surface, there was a marked increase in the incorporation of radiolabeled nucleosides per cell which appeared to be cell-density dependent. The predominant species group in developing dental plaque films during density-dependent growth was the sanguis streptococci; however, most other species present showed similar patterns of increased DNA synthesis as the density noted above approached 2.5 x 10(6) to 4.0 x 10(6) cells per mm2.
VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells
Authors: Sundberg C , Meek L , Carroll K , Das A , Ream W
Published: November 2016

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.
Pyrroloquinoline quinone, a chemotactic attractant for Escherichia coli
Authors: de Jonge R , Teixeira de Mattos M J , Stock J B , Neijssel O M
Published: November 2016

Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.
Analysis of the Erwinia chrysanthemi ferrichrysobactin receptor gene: resemblance to the Escherichia coli fepA-fes bidirectional promoter region and homology with hydroxamate receptors
Authors: Sauvage C , Franza T , Expert D
Published: November 2016

The fct cbsCEBA operon from the Erwinia chrysanthemi 3937 chrysobactin-dependent iron assimilation system codes for transport and biosynthetic functions. The sequence of the fct outer membrane receptor gene was determined. The fct promoter region displays a strong resemblance to the Escherichia coli bidirectional intercistronic region controlling the expression of the fepA-entD and fes-entF operons. An apparent Fur-binding site was shown to confer iron regulation on an fct::lac fusion expressed on a low-copy-number plasmid in a Fur-proficient E. coli strain. The fct gene consists of an open reading frame encoding a 735-amino-acid polypeptide with a signal sequence of 38 residues. The Fct protein has 36% sequence homology with the E. coli ferrichrome receptor FhuA and the Yersinia enterocolitica ferrioxamine receptor FoxA. On the basis of secondary-structure predictions and these homologies, we propose a two-dimensional folding model for Fct.
Random and directed mutagenesis to elucidate the functional importance of helix II and F-989 in the C-terminal secretion signal of Escherichia coli hemolysin
Authors: Chervaux C , Holland I B
Published: November 2016

The HlyA secretion signal sequence of approximately 46 residues is predicted to contain helix I and an amphipathic helix II separated by a short loop including the conserved Phe residue, F-989. All nine substitutions of Phe-989 drastically reduce secretion of HlyA. Directed mutagenesis identified a functional hot spot, EISK, in helix II. However, genetic analysis did not provide strong support for a functional helix II; rather, the results emphasized that individual residues, for example, E-978 and F-989, are essential irrespective of a specific secondary structure.
Genetic evidence for an activator required for induction of colicin-like bacteriocin 28b production in Serratia marcescens by DNA-damaging agents
Authors: Ferrer S , Viejo M B , Guasch J F , Enfedaque J , Regué M
Published: November 2016

Bacteriocin 28b production is induced by mitomycin in wild-type Serratia marcescens 2170 but not in Escherichia coli harboring the bacteriocin 28b structural gene (bss). Studies with a bss-lacZ transcriptional fusion showed that mitomycin increased the level of bss gene transcription in S. marcescens but not in the E. coli background. A S. marcescens Tn5 insertion mutant was obtained (S. marcescens 2170 reg::Tn5) whose bacteriocin 28b production and bss gene transcription were not increased by mitomycin treatment. Cloning and DNA sequencing of the mutated region showed that the Tn5 insertion was flanked by an SOS box sequence and three genes that are probably cotranscribed (regA, regB, and regC). These three genes had homology to phage holins, phage lysozymes, and the Ogr transcriptional activator of P2 and related bacteriophages, respectively. Recombinant plasmid containing this wild-type DNA region complemented the reg::Tn5 regulatory mutant. A transcriptional fusion between a 157-bp DNA fragment, containing the apparent SOS box upstream of the regA gene, and the cat gene showed increased chloramphenicol acetyltransferase activity upon mitomycin treatment. Upstream of the bss gene, a sequence similar to the consensus sequence proposed to bind Ogr protein was found, but no sequence similar to an SOS box was detected. Our results suggest that transcriptional induction of bacteriocin 28b upon mitomycin treatment is mediated by the regC gene whose own transcription would be LexA dependent.
The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain
Authors: Pedersen L B , Birkelund S , Holm A , Ostergaard S , Christiansen G
Published: November 2016

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1.
Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli
Authors: Eichler K , Buchet A , Lemke R , Kleber H P , Mandrand-Berthelot M A
Published: November 2016

Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis. When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene. The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon. This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries. In vivo overexpression consistently led to the synthesis of a 16-kDa protein. The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine. Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon. It was preceded by a cyclic AMP receptor protein motif centered at position -41.5. Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine. This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism. Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway. Expression of a caiF-lacZ operon fusion was subject to FNR regulator-mediated anaerobic induction and cyclic AMP receptor protein activation. The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors. Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism.
A novel dipeptidyl aminopeptidase from Pseudomonas sp. strain WO24
Authors: Ogasawara W , Ochiai K , Ando K , Yano K , Yamasaki M , Okada H , Morikawa Y
Published: November 2016

An activity similar to that of dipeptidyl aminopeptidase I (DAP I) which releases dipeptide from Gly-Arg-p-nitroanilide (Gly-Arg-pNA) was detected in a Pseudomonas sp. An enzyme was isolated and purified about 400-fold by a series of column chromatographies. The enzyme, named DAP BI (DAP from bacteria, type I), was revealed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass was estimated to be 82 kDa by SDS-PAGE and 65 kDa by gel filtration, suggesting that the enzyme may be a monomer. The enzyme had an isoelectric point of 4.7. It is optimally active at pH 9.0. The Km and Vmax of the enzyme for Gly-Arg-pNA were 0.25 mM and 195 micromol/min/mg, respectively. The purified enzyme did not hydrolyze Gly-Phe-pNA, which was also a substrate for DAP I, whereas it hydrolyzed Arg-Arg-4-methoxy-beta-naphthylamide (Arg-Arg-MNA), a model substrate for DAP III. The Km and Vmax for Arg-Arg-MNA were 0.019 mM and 145 micromol/min/mg, respectively. This purified enzyme can also catalyze the removal of Asp-Arg from the N termini of angiotensins I and II. The enzyme activity was completely inhibited by Zn(II) (0.5 mM), tosyl-L-Lys-chloromethyl ketone (0.1 mM), and leupeptin (0.1 mM) and partially inhibited by Co(II) (0.5 mM) and chymostatin (0.1 mM), whereas the enzyme was not affected by general serine protease inhibitors (phenylmethylsulfonyl fluoride and diisopropylfluorophosphate) and thiol protease inhibitors. The substrate specificity, classification of catalytic site, and other enzymatic properties demonstrate that this enzyme is distinct from the previously described mammalian DAPs I and III and Saccharomyces cerevisiae DAP III. These results indicate that DAP BI may be a new type of the DAP family.
A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli
Authors: Marykwas D L , Berg H C
Published: November 2016

The motor that drives the flagellar filament of Escherichia coli contains three "switch" proteins (FliG, FliM, and FliN) that together determine the direction of rotation. Each is required, in addition, for flagellar assembly and for torque generation. These proteins interact in the Saccharomyces cerevisiae two-hybrid system: FliG interacts with FliM, FliM interacts with itself, and FliM interacts with FliN. The interaction between FliG and FliM has been subjected to mutational analysis. FliG (fused to the GAL4 DNA-binding domain) and FliM (fused to a GAL4 transcription activation domain) together activate transcription of a GAL4-dependent lacZ reporter gene. DNA encoding FliG was mutagenized by error-prone amplification with Taq polymerase, mutant fliG genes were cloned (as DNA-binding domain-fliG gene fusions) in S. cerevisiae by gap repair of plasmid DNA, and mutants exhibiting an interaction defect were isolated in a two-hybrid screen. The mutations were each mapped to the first, second, or last third of the fliG gene by multifragment cloning in vivo and then identified by DNA sequencing. In this way, we identified 18 interaction-defective and 15 silent (non-interaction-defective) fliG mutations. Several residues within the middle third of FliG are strongly involved in the FliG-FliM interaction, while residues near the N or C terminus are less important. This clustering, when compared with results of previous studies, suggests that the FliG-FliM interaction plays a central role in switching.
Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri
Authors: Tallant T C , Krzycki J A
Published: November 2016

Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa corrinoid protein previously identified by its methylation following inhibition of methyl-CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha subunit is responsible for the activity. The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated corrinoid bound to the beta subunit of the 480-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high-pressure liquid chromatography with indirect photometric detection. Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cofactor binding subunit during isolation.
Regulation of VanB-type vancomycin resistance gene expression by the VanS(B)-VanR (B) two-component regulatory system in Enterococcus faecalis V583
Authors: Evers S , Courvalin P
Published: November 2016

Acquired VanA- and VanB-type glycopeptide resistance in enterococci is due to synthesis of modified peptidoglycan precursors terminating in D-lactate. As opposed to VanA-type strains which are resistant to both vancomycin and teicoplanin, VanB-type strains remain teicoplanin susceptible. We have determined the sequence of a 7,160-bp DNA fragment associated with VanB-type resistance in Enterococcus faecalis V583 that contains seven open reading frames. The distal part encoded the VanH (B), VanB, and VanX (B) proteins that are highly similar to the putative VanH, VanA, and VanX proteins responsible for VanA-type resistance. Upstream from the structural genes for these proteins were the vanY(B) gene encoding a D,D-carboxypeptidase and an open reading frame vanW with an unknown function. The proximal part of the gene cluster coded for the apparent VanS(B)-VanR (B) two-component regulatory system. VanR (B) was related to response regulators of the OmpR subclass, and VanS (B) was related to membrane-associated histidine protein kinases. Analysis of transcriptional fusions with a reporter gene and promoter mapping indicated that the VanR B-VanS B two-component regulatory system activates a promoter located immediately downstream from the vanS B gene. Vancomycin, but not teicoplanin, was an inducer, which explains teicoplanin susceptibility of VanB-type enterococci.
Flagellin A is essential for the virulence of Vibrio anguillarum
Authors: Milton D L , O'Toole R , Horstedt P , Wolf-Watz H
Published: November 2016

A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.