bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation and dephosphorylation of the NarQ, NarX, and NarL proteins of the nitrate-dependent two-component regulatory system of Escherichia coli
Authors: Schröder I , Wolin C D , Cavicchioli R , Gunsalus R P
Published: November 2016

The NarX, NarQ, and NarL proteins make up a nitrate-responsive regulatory system responsible for control of the anaerobic respiratory pathway genes in Escherichia coli, including nitrate reductase (narGHJI), dimethyl sulfoxide/trimethylamine-N-oxide reductase (dmsABC), and fumarate reductase (frdABCD) operons among others. The two membrane-bound proteins NarX and NarQ can independently sense the presence of nitrate and transfer this signal to the DNA-binding regulatory protein NarL, which controls gene expression by transcriptional activation or repression. To establish the role of protein phosphorylation in this process and to determine whether the NarX and NarQ proteins differ in their interaction with NarL, the cytoplasmic domains of NarX and NarQ were overproduced and purified. Both proteins were autophosphorylated in the presence of [gamma-32P]ATP and MgCl2 but not with [alpha-32P]ATP. Whereas these autophosphorylation reactions were unaffected by the presence of nitrate, molybdate, GTP, or AMP, ADP was an inhibitor. The phosphorylated forms of 'NarX and 'NarQ were stable for hours at room temperature. Each protein transferred its phosphoryl group to purified NarL protein, although 'NarQ-phosphate catalyzed the transfer reaction at an apparently much faster rate than did 'NarX-phosphate. In addition, NarL was autophosphorylated with acetyl phosphate but not with ATP as a substrate. NarL-phosphate remained phosphorylated for at least 3 h. However, addition of 'NarX resulted in rapid dephosphorylation of NarL-phosphate. In contrast, 'NarQ exhibited a much slower phosphatase activity with NarL-phosphate. These studies establish that the cytoplasmic domains of the two nitrate sensors 'NarX and 'NarQ differ in their ability to interact with NarL.
A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density
Authors: Baldwin W W , Kirkish M A , Koch A L
Published: November 2016

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.
micF antisense RNA has a major role in osmoregulation of OmpF in Escherichia coli
Authors: Ramani N , Hedeshian M , Freundlich M
Published: November 2016

micF RNA, produced from a multicopy plasmid, was originally shown to be a major factor in negative osmoregulation of the OmpF outer membrane protein in Escherichia coli. However, subsequent experiments with a micF deletion strain suggested that chromosomal micF RNA was not a key component in this process. We report here that micF RNA is essential for the reduction in OmpF levels in cells grown in media of low-to-intermediate levels of osmolarity. Under these conditions, the amount of OmpF was reduced up to 60% in the parent strain while OmpF levels were not altered in the micF deletion mutant. In medium of higher osmolarity, OmpF synthesis was strongly inhibited in both strains. RNA measurements showed that micF RNA levels rose rapidly in cells grown in low-to-intermediate levels of osmolarity concomitant with the reduction in OmpF protein, while ompF mRNA decreased strongly only during high-osmolarity conditions. Taken together, these results strongly suggest that the negative osmoregulation of OmpF at low-to-intermediate osmolarity levels requires micF RNA and that this is masked at higher osmolarity by the known strong inhibition of OmpF transcription by OmpR. Results consistent with this model were also obtained by using procaine, a compound reported to inhibit ompF expression by a mechanism very similar to that involved in osmoregulation.
Phylogenetic analysis of sequences from diverse bacteria with homology to the Escherichia coli rho gene
Authors: Opperman T , Richardson J P
Published: November 2016

Genes from Pseudomonas fluorescens, Chromatium vinosum, Micrococcus luteus, Deinococcus radiodurans, and Thermotoga maritima with homology to the Escherichia coli rho gene were cloned and sequenced, and their sequences were compared with other available sequences. The species for all of the compared sequences are members of five bacterial phyla, including Thermotogales, the most deeply diverged phylum. This suggests that a rho-like gene is ubiquitous in the Bacteria and was present in their common ancestor. The comparative analysis revealed that the Rho homologs are highly conserved, exhibiting a minimum identity of 50% of their amino acid residues in pairwise comparisons. The ATP-binding domain had a particularly high degree of conservation, consisting of some blocks with sequences of residues that are very similar to segments of the alpha and beta subunits of F1-ATPase and of other blocks with sequences that are unique to Rho. The RNA-binding domain is more diverged than the ATP-binding domain. However, one of its most highly conserved segments includes a RNP1-like sequence, which is known to be involved in RNA binding. Overall, the degree of similarity is lowest in the first 50 residues (the first half of the RNA-binding domain), in the putative connector region between the RNA-binding and the ATP-binding domains, and in the last 50 residues of the polypeptide. Since functionally defective mutants for E. coli Rho exist in all three of these segments, they represent important parts of Rho that have undergone adaptive evolution.
Cross-regulation by XylR and DmpR activators of Pseudomonas putida suggests that transcriptional control of biodegradative operons evolves independently of catabolic genes
Authors: Fernández S , Shingler V , De Lorenzo V
Published: November 2016

The Pu promoter of the toluene degradation plasmid pWW0 of Pseudomonas putida drives expression of an operon involved in the sequential oxidation of toluene and m- and p-xylenes to benzoate and toluates, respectively. Similarly, the Po promoter of plasmid pVI150 controls expression of an operon of Pseudomonas sp. strain CF600 which is required for the complete catabolism of phenol and cresols. These promoters, which both belong to the sigma 54-dependent class, are regulated by their cognate activators, XylR and DmpR, respectively. XylR and DmpR are homologous proteins, and both require aromatic compounds as effector molecules for activity. However, these two proteins respond to different profiles of aromatic compounds. The activity of each promoter in the presence of the heterologous regulator was monitored using lacZ and luxAB reporter systems. Genetic evidence is presented that the two activators can functionally substitute each other in the regulation of their corresponding promoters by binding the same upstream DNA segment. Furthermore, when coexpressed, the two proteins appear to act simultaneously on each of the promoters, expanding the responsiveness of these systems to the presence of effectors of both proteins. Potential mechanisms for the occurrence of evolutionary divergence between XylR and DmpR are discussed in view of the DNA sequence similarities among Pu, Po, and a third XylR-responsive promoter, Ps.
Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al
Authors: Goldman B S , Lin J T , Stewart V
Published: November 2016

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilatory pathway. The structural genes for assimilatory nitrate and nitrite reductases together with genes necessary for nitrate transport form an operon, nasFEDCBA. Expression of the nasF operon is regulated both by general nitrogen control and also by nitrate or nitrite induction. We have identified a gene, nasR, that is necessary for nitrate and nitrite induction. The nasR gene, located immediately upstream of the nasFEDCBA operon, encodes a 44-kDa protein. The NasR protein shares carboxyl-terminal sequence similarity with the AmiR protein of Pseudomonas aeruginosa, the positive regulator of amiE (aliphatic amidase) gene expression. In addition, we present evidence that the nasF operon is not autogenously regulated.
Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates
Authors: Death A , Ferenci T
Published: November 2016

Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.
Vibrio cholerae fur mutations associated with loss of repressor activity: implications for the structural-functional relationships of fur
Authors: Lam M S , Litwin C M , Carroll P A , Calderwood S B
Published: November 2016

We used the Vibrio cholerae Fur protein as a model of iron-sensitive repressor proteins in gram-negative bacteria. Utilizing manganese mutagenesis, we isolated twelve independent mutations in V. cholerae fur that resulted in partial or complete loss of Fur repressor function. The mutant fur genes were recovered by PCR and sequenced; 11 of the 12 contained point mutations (two of which were identical), and one contained a 7-bp insertion that resulted in premature truncation of Fur. All of the mutants, except that containing the prematurely truncated Fur, produced protein by Western blot (immunoblot) analysis, although several had substantially smaller amounts of Fur and two made an immunoreactive protein that migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nine of the 11 point mutations altered amino acids that are identical in all of the fur genes sequenced so far, suggesting that these amino acids may play important structural or functional roles in Fur activity. Eight of the point mutations occurred in the amino-terminal half of Fur, which is thought to mediate DNA binding; most of these mutations occurred in conserved amino acids that have been previously suggested to play a role in the interaction between adjacent alpha-helices of the protein. Three of the point mutations occurred in the carboxy-terminal half of Fur, which is thought to bind iron. One mutation at histidine-90 was associated with complete loss of Fur function; this amino acid is within a motif previously suggested as being involved in iron binding by Fur. The fur allele mutant at histidine-90 interfered with iron regulation by wild-type fur in the same cell when the mutant allele was present at higher copy number; wild-type fur was dominant over all other fur mutant alleles studied. These results are analyzed with respect to previous models of the structure and function of Fur as an iron-sensitive repressor.
Nucleotide sequence of the Wolinella succinogenes flagellin, which contains in the antigenic domain two conserved regions also present in Campylobacter spp. and Helicobacter pylori
Authors: Schuster S C , Bauer M , Kellermann J , Lottspeich F , Baeuerlein E
Published: November 2016

Wolinella succinogenes possesses one polar flagellum, which shows a characteristic surface pattern of parallel lines along the axis of the filament in electron microscopic images. We determined the gene sequence of the Wolinella flagellin, which is, as in most other bacteria, the only structural component of the filament. Sequence comparison with other members of the Proteobacteria revealed two highly conserved regions in the central part of the flagellin molecule among Campylobacter spp. and Helicobacter pylori, an area that had previously been described as highly variable. Similar surface patterns are found in related polarly flagellated bacteria, but not in Escherichia coli and Bacillus subtilis, which also lack these conserved regions.
The F- or V-type Na(+)-ATPase of the thermophilic bacterium Clostridium fervidus
Authors: Speelmans G , Poolman B , Abee T , Konings W N
Published: November 2016

Clostridium fervidus is a thermophilic, anaerobic bacterium which uses solely Na+ as a coupling ion for energy transduction. Important features of the primary Na+ pump (ATPase) that generates the sodium motive force are presented. The advantage of using a sodium rather than a proton motive force at high temperatures becomes apparent from the effect of temperature on H+ and Na+ permeation in liposomes.
A new type of NtrC transcriptional activator
Authors: Foster-Hartnett D , Cullen P J , Monika E M , Kranz R G
Published: November 2016

The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for activation, and (iii) they activate the sigma 54 RNA polymerase. We have characterized the NtrC protein from a photosynthetic bacterium, Rhodobacter capsulatus, which represents a metabolically versatile group of bacteria found in aquatic environments. We have shown that the R. capsulatus NtrC protein (RcNtrC) binds to two tandem sites that are distant from promoters that it activates, nifA1 and nifA2. These tandem binding sites are shown to be important for RcNtrC-dependent nitrogen regulation in vivo. Moreover, the conserved nucleotide-binding fold of RcNtrC is required to activate nifA1 and nifA2 but is not required for DNA binding of RcNtrC to upstream activation sequences. However, nifA1 and nifA2 genes do not require the sigma 54 for activation and do not contain the highly conserved nucleotides that are present in all sigma 54-type, EBP-activated promoters. Thus, the NtrC from this photosynthetic bacterium represents a novel member of the class of bacterial EBPs. It is probable that this class of EBPs is more versatile in prokaryotes than previously envisioned.
Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17
Authors: Wodara C , Kostka S , Egert M , Kelly D P , Friedrich C G
Published: November 2016

The coding region for lithotrophic sulfur oxidation (Sox) in Paracoccus denitrificans GB17 was identified by isolation of a transposon Tn5-mob mutant with a Sox- phenotype (strain TP19). The corresponding wild-type region was cloned previously (G. Mittenhuber, K. Sonomoto, M. Egert, and C. G. Friedrich, J. Bacteriol. 173:7340-7344, 1991). Sequence analysis of a 2.5-kb subclone that complemented strain TP19 revealed that Tn5-mob was inserted into a coding region for a 553-amino-acid polypeptide named SoxB. This polypeptide had an M(r) of 60.573, including a possible signal peptide. The function of the SoxB protein of P. denitrificans GB17 appeared to be identical to that of enzyme B of the thiosulfate-oxidizing enzyme system of Thiobacillus versutus. The amino acid compositions of the two proteins were identical, and the amino acid sequences of three internal peptides of enzyme B as determined by Edman degradation were identical to corresponding sequences of the deduced SoxB protein of P. denitrificans GB17.
Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation
Authors: Escarceller M , Hicks J , Gudmundsson G , Trump G , Touati D , Lovett S , Foster P L , McEntee K , Goodman M F
Published: November 2016

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.
Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation
Authors: Gu Z , Harrod R , Rogers E J , Lovett P S
Published: November 2016

Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA.
The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity
Authors: Chen B J , Carroll P , Samson L
Published: November 2016

Escherichia coli can ameliorate the toxic effects of alkylating agents either by preventing DNA alkylation or by repairing DNA alkylation damage. The alkylation-sensitive phenotype of E. coli alkB mutants marks the alkB pathway as an extremely effective defense mechanism against the cytotoxic effects of the SN2, but not the SN1, alkylating agents. Although it is clear that AlkB helps cells to better handle alkylated DNA, no DNA alkylation repair function could be assigned to the purified AlkB protein, suggesting that AlkB either acts as part of a complex or acts to regulate the expression of other genes whose products are directly responsible for alkylation resistance. However, here we present evidence that the provision of alkylation resistance is an intrinsic function of the AlkB protein per se. We expressed the E. coli AlkB protein in two human cell lines and found that it confers the same characteristic alkylation-resistant phenotype in this foreign environment as it does in E. coli. AlkB expression rendered human cells extremely resistant to cell killing by the SN2 but not the SN1 alkylating agents but did not affect the ability of dimethyl sulfate (an SN2 agent) to alkylate the genome. We infer that SN2 agents produce a class of DNA damage that is not efficiently produced by SN1 agents and that AlkB somehow prevents this damage from killing the cell.
Dual regulation of inaA by the multiple antibiotic resistance (mar) and superoxide (soxRS) stress response systems of Escherichia coli
Authors: Rosner J L , Slonczewski J L
Published: November 2016

The roles of the marRAB (multiple antibiotic resistance) operon and soxRS (superoxide response) genes in the regulation of inaA, an unlinked weak-acid-inducible gene, were studied. inaA expression was estimated from the beta-galactosidase activity of a chromosomal inaA1::lacZ transcriptional fusion. marR mutations that elevate marRAB transcription and engender multiple antibiotic resistance elevated inaA expression by 10- to 20-fold over that of the wild-type. Similarly, one class of inaA constitutive mutants that mapped to the mar region were multiply antibiotic resistant. Overexpression of marA alone on a multicopy plasmid caused high constitutive expression of inaA in a strain with an extensive (39-kbp) marRAB deletion. Salicylate, an inducer of marRAB and of an unidentified mar-independent antibiotic resistance system, induced inaA by 6-fold. A portion of this induction was also mar independent. Two soxRS constitutive mutants that were tested showed elevated levels of inaA. Paraquat, an inducer of the soxRS system, elevated inaA expression by 6- to 9-fold. This induction was soxRS dependent and not mar dependent, whereas induction of inaA by salicylate was not dependent on soxRS. Paraquat induced resistance to norfloxacin in the mar-deleted strain but not in a soxRS-deleted strain. Thus, induction of multiple antibiotic resistance and inaA by salicylate occurs via mar and an unidentified pathway, while induction by paraquat occurs via soxRS.
Reciprocal light-dark transcriptional control of nif and rbc expression and light-dependent posttranslational control of nitrogenase activity in Synechococcus sp. strain RF-1
Authors: Chow T J , Tabita F R
Published: November 2016

Synechococcus sp. strain RF-1 exhibits a circadian rhythm of N2 fixation when cells are grown under a light-dark cycle, with nitrogenase activity observed only during the dark period. This dark-dependent activity correlated with nif gene transcription in strain RF-1. By using antibodies against dinitrogenase reductase (the Fe protein of the nitrogenase complex), it was found that there was a distinct shift in the mobility of this protein on sodium dodecyl sulfate gels during the light-dark cycle. The Fe protein was present only when cells were incubated in the dark. Upon illumination, there was a conversion of all Fe protein to a modified form, after which it rapidly disappeared from extracts. These studies indicated that all nitrogenase activity present during the dark cycle resulted from de novo synthesis of nitrogenase. Upon entering the light phase, cells appeared to quickly degrade the modified form of Fe protein, perhaps as a result of activating or inducing a protease. By contrast, transcription of the rbcL gene, which encodes the catalytic subunit of the key enzyme of CO2 fixation (a light-dependent process), was enhanced in the light.
Maturation and localization of the TolB protein required for colicin import
Authors: Isnard M , Rigal A , Lazzaroni J C , Lazdunski C , Lloubes R
Published: November 2016

The tolB gene has been shown previously to encode two proteins of 47.5 kDa (TolB) and 43 kDa (TolB*). To explain the presence of these two forms, two hypotheses have been proposed: TolB might be posttranslationally processed to TolB*, or an internal in-frame translation initiation resulting in TolB* may occur (S. K. Levengood and R. E. Webster, J. Bacteriol. 171:6600-6609, 1989). To address this question, TolB was tagged by inserting in its C-terminal region an epitope recognized by monoclonal antibody 1C11 without altering the function of TolB. It was then demonstrated that the functional protein corresponded to TolB*, the mature periplasmic protein, and that TolB was its precursor form, which was observed only when the protein was overexpressed. These two forms were purified by immunoprecipitation, and their N-terminal sequences were determined. An antibody directed against TolB was raised, which confirmed the results obtained with the tagged TolB.
Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Floriano B , Herrero A , Flores E
Published: November 2016

A cloned DNA fragment from Anabaena sp. strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD. The expression of Anabaena sp. strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level. Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine. Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E. coli RNA polymerase sigma 70 consensus promoter. A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures.
Additive effect of tolC and rfa mutations on the hydrophobic barrier of the outer membrane of Escherichia coli K-12
Authors: Fralick J A , Burns-Keliher L L
Published: November 2016

Studies using tolC mutant derivatives of deep rough (rfa) mutants indicate that tolC and rfa mutations have an additive effect with respect to their sensitivity to hydrophobic agents, suggesting that they are not acting through a mutual mechanism to alter the permeability of the outer membrane.
Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements
Authors: Sokol P A , Luan M Z , Storey D G , Thirukkumaran P
Published: November 2016

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.
A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis
Authors: Fields K A , Plano G V , Straley S C
Published: November 2016

The causative agent of plague, Yersinia pestis, contains a 75-kb plasmid, pCD1, which carries a virulence-related stimulon called the low-Ca2+ response stimulon (LCRS). LCRS operons are regulated by the environmental signals of temperature and Ca2+. This study characterized a portion of the lcrB region of pCD1, known to contain at least one gene necessary for the regulation of LCRS operons by Ca2+. The sequence of a 2-kb region revealed three open reading frames, designated yscQ, yscR, and yscS, predicted to encode acidic proteins of 34.4, 24.4, and 8.5 kDa. All three proteins were homologous to proteins involved in flagellar function or virulence. An antipeptide antibody specific for YscR was used to localize YscR to the inner membrane of Y. pestis. Analysis of yscR-phoA fusions supported a model for yscR which predicts four transmembrane regions and a large, central hydrophilic domain. In-frame deletion mutations of yscQ and yscR were constructed and moved into Y. pestis. Both mutants failed to show the restriction of growth that normally accompanies maximal LCRS induction. Unlike the parent Y. pestis, the yscR mutant did not respond to the absence of Ca2+ by increasing the net transcription or translation of the LCRS-encoded V antigen, YopM, or LcrG. The yscR mutant also was defective for secretion of V antigen, YopM, and LcrG. These findings implicate a dual role for YscR in regulation of LCRS operons and secretion of LCRS proteins and add to the developing picture of how secretion of virulence proteins may be coupled to transcriptional regulation in yersiniae.
Regulation of alpha- and beta-hemolysins by the sar locus of Staphylococcus aureus
Authors: Cheung A L , Ying P
Published: November 2016

We recently identified a locus on the Staphylococcus aureus chromosome, designated sar, for staphylococcal accessory regulator, that is involved in the global regulation of extracellular and cell wall-associated proteins. Previous phenotypic and Southern blot analyses with Tn917 and agr probes indicated that this locus is distinct from agr, a previously described global regulator of exoproteins in S. aureus. To understand the mode of regulatory control of exoprotein synthesis by the sar locus, the sar genotype was transduced from the original sar mutant 11D2 into two prototypic S. aureus strains, RN6390 and RN450, with well-defined genetic backgrounds. An analysis of extracellular protein profiles by use of silver-stained sodium dodecyl sulfate gels revealed alterations in the pattern of exoprotein production in the late log-early stationary phase in the sar mutants in comparison with the corresponding parents. In addition, most of the phenotypic changes that occurred in the conversion from the sar+ genotype to the sar genotype in mutant 11D2 were also found in these mutants. Northern (RNA) blot analyses of two exoprotein transcripts (alpha- and beta-hemolysins) from strain RN6390 and its corresponding sar mutant revealed downregulation of these transcripts in the mutant. Serial studies of these hemolysin transcripts at various growth intervals demonstrated that the transcriptional regulation of the hemolysin genes by the sar locus began during the log phase and continued into the postexponential phase. These data suggested that the sar locus probably regulates exoprotein genes at the transcriptional level. This mode of regulation is similar to that of exoprotein target gene transcription by agr.
Lactobacillus plantarum ldhL gene: overexpression and deletion
Authors: Ferain T , Garmyn D , Bernard N , Hols P , Delcour J
Published: November 2016

Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.
The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment
Authors: Brandner J P , Donohue T J
Published: November 2016

Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a member of the heme-containing cytochrome c protein family that is found in the periplasmic space of this gram-negative bacterium. This exported polypeptide is made as a higher-molecular-weight precursor with a typical procaryotic signal peptide. Therefore, cyt c2 maturation is normally expected to involve precursor translocation across the cytoplasmic membrane, cleavage of the signal peptide, and covalent heme attachment. Surprisingly, synthesis as a precursor polypeptide is not a prerequisite for cyt c2 maturation because deleting the entire signal peptide does not prevent export, heme attachment, or function. Although cytochrome levels were reduced about threefold in cells containing this mutant protein, steady-state cyt c2 levels were significantly higher than those of other exported bacterial polypeptides which contain analogous signal peptide deletions. Thus, this mutant protein has the unique ability to be translocated across the cytoplasmic membrane in the absence of a signal peptide. The covalent association of heme with this mutant protein also suggests that the signal peptide is not required for ligand attachment to the polypeptide chain. These results have uncovered some novel aspects of bacterial c-type cytochrome biosynthesis.
nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum
Authors: Stacey G , Luka S , Sanjuan J , Banfalvi Z , Nieuwkoop A J , Chun J Y , Forsberg L S , Carlson R
Published: November 2016

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.
Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358
Authors: de Groot A , Heijnen I , de Cock H , Filloux A , Tommassen J
Published: November 2016

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.
A new gene required for cellulose production and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon
Authors: Standal R , Iversen T G , Coucheron D H , Fjaervik E , Blatny J M , Valla S
Published: November 2016

Recently, it was shown that a cellulose-negative mutant (Cel1) of Acetobacter xylinum ATCC 23769 carried an insertion of an indigenous transposable element (IS1031A) about 500 bp upstream of the bcs operon, required for cellulose synthesis. Here we show that Cel1 can be complemented by wild-type DNA covering the insertion point. Nucleotide sequencing of this region revealed the presence of two open reading frames, ORF1 and ORF2. ORF2, which is disrupted by the IS1031A insertion in Cel1, potentially encodes the complementing function. ORF1 encodes a protein (CMCax) with significant homology to previously described endoglucanases. A cloned DNA fragment containing ORF1 expressed a carboxymethyl cellulose-hydrolyzing activity in Escherichia coli. In A. xylinum, CMCax is secreted into the culture growth medium. The CMCax mature protein consists of 322 amino acids and has a molecular mass of 35.6 kDa.
Involvement of phospholipid end groups of group C Neisseria meningitidis and Haemophilus influenzae type b polysaccharides in association with isolated outer membranes and in immunoassays
Authors: Arakere G , Lee A L , Frasch C E
Published: November 2016

There are several bacterial polysaccharides (PSs) which contain a terminal lipid moiety. It has been postulated that these terminal lipid moieties anchor the PSs to the outer membrane of the bacteria. Our studies have shown that incubation of native PS from group C Neisseria meningitidis or Haemophilus influenzae type b with isolated outer membrane vesicles results in association of a portion of the PS with the vesicles. Removal of the terminal lipid from the PS by treatment with phospholipase A2 or phospholipase D eliminates this association. In other studies, it was shown that delipidated PSs are not suitable as solid-phase antigens in a currently used enzyme-linked immunosorbent assay (ELISA). Measurement of antibody units in the reference sera by using delipidated PSs as antigens in an ELISA yielded negligible absorbance compared with native PSs when methylated human serum albumin was used to coat the PSs to the plate. Nevertheless, phospholipase A2 and phospholipase D treatment did not noticeably affect antigenic epitopes, since soluble group C PS without the terminal lipid bound antibody as effectively as the native PS did, as measured by a competitive inhibition assay. Both hydrophobic and electrostatic interactions are important for the binding of group C N. meningitidis PS to the ELISA plate, while charge interactions seem to be sufficient for binding the more negatively charged H. influenzae type b PS.
Inhibition of erythromycin synthesis by disruption of malonyl-coenzyme A decarboxylase gene eryM in Saccharopolyspora erythraea
Authors: Hsieh Y J , Kolattukudy P E
Published: November 2016

Malonyl-coenzyme A (malonyl-CoA) decarboxylase is widely distributed in prokaryotes and eukaryotes. However, the biological function of this enzyme has not been established in any organism. To elucidate the structure and function of this enzyme, the malonyl-CoA decarboxylase gene from Saccharopolyspora erythraea (formerly Streptomyces erythreaus) was cloned and sequenced. This gene would encode a polypeptide of 417 amino acids. The deduced amino acid sequence matched the experimentally determined amino acid sequences of 25 N-terminal residues each of the enzyme and of an internal peptide obtained by proteolysis of the purified enzyme. This decarboxylase showed homology with aminoglycoside N6'-acetyltransferases of Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella pneumoniae. Northern (RNA) blot analysis revealed a single transcript. The transcription initiation site was 220 bp upstream of the start codon. When expressed in Escherichia coli, the S. erythraea malonyl-CoA decarboxylase gene yielded a protein that cross-reacted with antiserum prepared against S. erythraea malonyl-CoA decarboxylase and catalyzed decarboxylation of [3-14C]malonyl-CoA to acetyl-CoA and 14CO2. The S. erythraea malonyl-CoA decarboxylase gene was disrupted by homologous recombination using an integrating vector pWHM3. The gene-disrupted transformant did not produce immunologically cross-reacting 45-kDa decarboxylase, lacked malonyl-CoA decarboxylase activity, and could not produce erythromycin. Exogenous propionate restored the ability to produce erythromycin. These results strongly suggest that the decarboxylase provides propionyl-CoA for erythromycin synthesis probably via decarboxylation of methylmalonyl-CoA derived from succinyl-CoA, and therefore the malonyl-CoA decarboxylase gene is designated eryM. The gene disrupted mutants also did not produce pigments.
Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria
Authors: Paster B J , Dewhirst F E , Olsen I , Fraser G J
Published: November 2016

The phylogenetic structure of the bacteroides subgroup of the cytophaga-flavobacter-bacteroides (CFB) phylum was examined by 16S rRNA sequence comparative analysis. Approximately 95% of the 16S rRNA sequence was determined for 36 representative strains of species of Prevotella, Bacteroides, and Porphyromonas and related species by a modified Sanger sequencing method. A phylogenetic tree was constructed from a corrected distance matrix by the neighbor-joining method, and the reliability of tree branching was established by bootstrap analysis. The bacteroides subgroup was divided primarily into three major phylogenetic clusters which contained most of the species examined. The first cluster, termed the prevotella cluster, was composed of 16 species of Prevotella, including P. melaninogenica, P. intermedia, P. nigrescens, and the ruminal species P. ruminicola. Two oral species, P. zoogleoformans and P. heparinolytica, which had been recently placed in the genus Prevotella, did not fall within the prevotella cluster. These two species and six species of Bacteroides, including the type species B. fragilis, formed the second cluster, termed the bacteroides cluster. The third cluster, termed the porphyromonas cluster, was divided into two subclusters. The first contained Porphyromonas gingivalis, P. endodontalis, P. asaccharolytica, P. circumdentaria, P. salivosa, [Bacteroides] levii (the brackets around genus are used to indicate that the species does not belong to the genus by the sensu stricto definition), and [Bacteroides] macacae, and the second subcluster contained [Bacteroides] forsythus and [Bacteroides] distasonis. [Bacteroides] splanchnicus fell just outside the three major clusters but still belonged within the bacteroides subgroup. With few exceptions, the 16 S rRNA data were in overall agreement with previously proposed reclassifications of species of Bacteroides, Prevotella, and Porphyromonas. Suggestions are made to accommodate those species which do not fit previous reclassification schemes.