Simultaneous production of two types of beta-lactamase in Escherichia coli and Providencia stuartii.

The production of beta-lactamase has been studied in two strains isolated from clinical samples: Providencia stuartii MULB 501 and Escherichia coli MULB 130. These strains were selected for their high resistance level to penicillins and cephalosporins. Determination and identification of beta-lactamase activity were achieved by combining several up-to-date methods including (i) neutralization by anti-beta-lactamase sera, (ii) purification by affinity chromatography on cephalosporin C linked Sepharose 4B, (iii) determination of substrate specificity and kinetic values (Km and Vmax) by a computerized microacidimetric method, and (iv) isoelectric focusing. The results clearly demonstrate that in these two strains there is a simultaneous production of different beta-lactamases: the first one is similar to the TEM penicillinase and the second one shares a typical cephalosporinase profile. This double beta-lactamase production is a relatively rare phenomenon.     

Characterization of aarA, a pleiotrophic negative regulator of the 2'-N-acetyltransferase in Providencia stuartii.

We have utilized transposon mutagenesis to obtain insertional mutations in Providencia stuartii that activate the chromosomal aac(2')-la gene. Two closely linked mini-Tn5Cm insertions were obtained in a locus designated aarA, and a single insertion was obtained in a separate locus, aarC. Nucleotide sequence analysis, complementation studies, and localization of the sites of mini-Tn5Cm insertion have allowed the identification of the aarA coding region. The deduced AarA protein had a molecular mass of 31,086 kDa and displayed characteristics of an integral membrane protein. A strain deleted for the aarA gene by allelic exchange showed at least a fourfold increase in the accumulation of aac(2')-la mRNA and an eightfold increase in aminoglycoside resistance. Mutations in aarA were pleiotrophic and also resulted in loss of pigmentation and a deficiency in cell separation during division.     

Termination of Deoxyribonucleic Acid in Escherichia coli by 2′,3′-Dideoxyadenosine

2′,3′-Dideoxyadenosine was previously shown to be lethal to Escherichia coli and to inhibit deoxyribonucleic acid (DNA) synthesis irreversibly in this organism. It was also shown that triphosphate of this analogue terminates DNA chains in an in vitro system. Data presented here show that the nucleoside is relatively insensitive to E. coli adenosine deaminase and is converted intracellularly into the dideoxynucleotide, including the triphosphate. Thymine nucleotide pools were not reduced in inhibited bacteria, nor did preformed DNA break down. Some adenine was liberated from the dideoxyadenosine on incubation, and the latter was incorporated into ribonucleic acid. Nevertheless, about 4,000 molecules of the dideoxynucleoside were incorporated into DNA per cell. The dideoxynucleotide occurred in DNA chains in a terminal position, liberated selectively by venom phosphodiesterase. The possible nature of the lethal event is discussed.     

Identification and characterization of a short 2′–3′ bond-forming ribozyme

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2′–3′ phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.     

Importance of a 5′ Stem-Loop for Longevity of papA mRNA in Escherichia coli

High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5' untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5' untranslated region, as evidenced by the significant acceleration in papA mRNA decay that results from its removal.     

Identification and Characterization of the Functional Elements within the Tobacco Etch Virus 5′ Leader Required for Cap-Independent Translation

Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m(7)GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5' leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5'-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5' end.     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Role of CysE in production of an extracellular signaling molecule in Providencia stuartii and Escherichia coli: loss of CysE enhances biofilm formation in Escherichia coli

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.     

Role of Cyclic Adenosine 3′,5′-Monophosphate in the In Vivo Expression of the Galactose Operon of Escherichia coli

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.     

Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.     

Phosphorolysis of 5-Fluoro-2′-Deoxyuridine in Escherichia coli and Its Inhibition by Nucleosides

The effect of ribonucleosides and deoxyribonucleosides on the phosphorolysis of 5-fluoro-2'-deoxyuridine (FUdR) was examined in cells of Escherichia coli. All nucleosides tested except guanosine and deoxyguanosine inhibited phosphorolysis. By using genetically marked strains it was found that in vivo FUdR is a specific substrate of thymidine phosphorylase.     

Requirement of Adenosine 3′,5′-Cyclic Phosphate for Formation of the Phage Lambda Receptor in Escherichia coli

Adenosine 3',5'-cyclic phosphate is indispensable for formation of the phage lambda receptor in Escherichia coli K-12.     

uspB, a New ςS-Regulated Gene in Escherichia coli Which Is Required for Stationary-Phase Resistance to Ethanol

The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to uspA, stationary-phase induction of uspB requires the sigma factor ς^S. The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering ς^S levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon’s previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.     

β-β′ Subunits of Ribonucleic Acid Polymerase in Episome-Free Minicells of Escherichia coli

Episome-free minicells of Escherichia coli, previously shown to lack ribonucleic acid polymerase activity, do contain the beta-beta' subunits of the polymerase.