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The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nif iscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nif iscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe-4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNA Arg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNA Arg s; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNA Arg s from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNA Arg ICG , the most abundant species of E. coli tRNA Arg s, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNA Arg ICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNA Arg ICG dominant-negatively impairs translation in vitro . Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNA Arg ICG , which is consistent with our structural model. Finally, elevation of cleaved tRNA Arg ICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNA Arg ICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops. Abbreviations: mnm 5 U: 5-methylaminomethyluridine; mcm 5 s 2 U: 5-methoxycarbonylmethyl-2-thiouridine.

Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.

A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.

RNAs, such as mRNA, rRNA and tRNA, are essential macromolecules for cell survival and maintenance. Any perturbation of these molecules, such as by degradation or mutation, can be toxic to cells and may occasionally induce cell death. Therefore, cells have mechanisms known as quality control systems to eliminate abnormal RNAs. Although tRNA is a stable molecule, the anticodon loop is quite susceptible to tRNA-targeting RNases such as colicin E5 and colicin D. However, the mechanism underlying cellular reaction to tRNA cleavage remains unclear. It had long been believed that tRNA cleavage by colicins E5 and D promptly induces cell death because colony formation of the sensitive cells is severely reduced; this indicates that cells do not resist the tRNA cleavage. Here, we show that Escherichia coli cells enter a bacteriostatic state against the tRNA cleavage of colicins D and E5. The bacteriostasis requires small protein B (SmpB) and transfer-messenger RNA (tmRNA), which are known to mediate trans-translation. Furthermore, another type of colicin, colicin E3 cleaving rRNA, immediately reduces the viability of sensitive cells. Moreover, nascent peptide degradation has an additive effect on bacteriostasis. Considering the recent observation that tRNA cleavage may be used as a means of cell-to-cell communication, tRNA cleavage could be used by bacteria not only to dominate other bacteria living in the same niche, but also to regulate growth of their own or other cells.

It has long been believed that amino acids comprising proteins of all living organisms are only of the L-configuration, except for Gly. However, peptidyl D-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of D-amino acids are naturally present in usual proteins. Thus we analyzed the D-amino acid contents of His-tag-purified beta-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110 degrees C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into D- and L-enantiomers. The contents of D-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index D/(D + L) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of D-amino acids were reproducibly detected, the D-amino acid profile being specific to an individual protein. This finding indicated the likelihood that D-amino acids are in fact present in the purified proteins. On the other hand, the D-amino acid contents of proteins were hardly influenced by the addition of D- or L-amino acids to the cultivation medium, whereas intracellular free D-amino acids sensitively varied according to the extracellular conditions. The origin of these D-amino acids detected in proteins was discussed.

The tRNase domain of colicin D, which cleaves only tRNA(Arg)s at the 3' side of their anticodon loops, has been expressed in Escherichia coli with its inhibitor protein and purified to a form free from the inhibitor using a low-pH buffer. Crystals were obtained by the hanging-drop vapour-diffusion method at 278 K from a buffer containing 100 mM Tris-HCl pH 8.5, 22% PEG MME 2000 and 1 mM nickel(II) chloride. Diffraction data to 1.05 A resolution were collected at BL41XU, SPring-8. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.7, b = 65.5, c = 96.5 A.

Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNA(Tyr), tRNA(His), tRNA(Asn) and tRNA(Asp). Since this activity is confined to its 115 amino acid long C-terminal domain (CRD), the recognition mechanism of E5-CRD is of great interest. The four tRNA substrates share the unique sequence UQU within their anticodon loops, and are cleaved between Q (modified base of G) and 3' U. Synthetic minihelix RNAs corresponding to the substrate tRNAs were completely susceptible to E5-CRD and were cleaved in the same manner as the authentic tRNAs. The specificity determinant for E5-CRD was YGUN at -1 to +3 of the 'anticodon'. The YGU is absolutely required and the extent of susceptibility of minihelices depends on N (third letter of the anticodon) in the order A > C > G > U accounting for the order of susceptibility tRNA(Tyr) > tRNA(Asp) > tRNA(His), tRNA(Asn). Contrastingly, we showed that GpUp is the minimal substrate strictly retaining specificity to E5-CRD. The effect of contiguous nucleotides is inconsistent between the loop and linear RNAs, suggesting that nucleotide extension on each side of GpUp introduces a structural constraint, which is reduced by a specific loop structure formation that includes a 5' pyrimidine and 3' A.

It is generally believed that only L-amino acids are acceptable in protein synthesis, though some D-amino acids, including D-tyrosine, D-aspartate, and D-tryptophan are known to be bound enzymatically to tRNAs. In this report, we newly show that D-histidine and D-lysine are also able to be the substrates of respective Escherichia coli aminoacyl-tRNA synthetases.

Background: The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA., Results: A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced., Conclusions: The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.

In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties. (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (rnh) conditions. (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E. coli chromosome and had Chi activities (H. Nishitani, M. Hidaka, and T. Horiuchi, Mol. Gen. Genet. 240:307-314, 1993). Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent. Other Hot groups showed termination-independent Hot activities. In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity. The HotA activity which had disappeared was reactivated by insertion of a new, properly oriented Chi sequence at the position between the HotA DNA and the TerB site. On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.

To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Kmr DNA fragment and transformed into an rnh- derivative host. From the Kmr transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed "Hot", derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.

To search for heretofore unidentified DNA replication termination (Ter) sites on the Escherichia coli chromosome, we screened the entire Kohara lambda bacteriophage library using as probes the four known 22-bp Ter sequences. We found a Ter site, which we named TerE, located at 23.2 min on the linkage map. TerE inhibits only counterclockwise DNA replication. Macroscopically, five Ter sites are located in a periodic arrangement on the genome.

Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.

Double-stranded cDNA was prepared from prorennin-specific mRNA by sequential actions of reverse transcriptase, DNA polymerase and S1 nuclease, and inserted into the Sa/I site of pBR322 by the poly(dG)-(dC) annealing method. Transformation of Escherichia coli C600 r- m- by the hybrid plasmid yielded transformants containing prorennin cDNA. The presence of the cDNA sequence in these clones was confirmed by both colony hybridization and hybrid-arrested translation of the mRNA in vitro. The largest size of the cloned cDNA was 1,020 bp.

A 4.32 kb DNA fragment, on which the DNA replication terminus (terR) site of plasmid R 6K was located, was inserted into the unique EcoRI site of plasmid pUC9. To detect replication intermediate molecules with a replication fork halted at the terR site, a cell DNA extract was digested with EcoRI, electrophoresed through an agarose gel and stained with ethidium bromide. In addition to two major bands, one derived from vector DNA and the other from the ter insert fragment, two extra minor bands were detected. Following DNA-DNA hybridization and electron microscopic observation we concluded that the two minor bands corresponded to the two Y-shaped molecules, produced from the theta-shaped intermediate molecules by EcoRI digestion.

Identification of the consensus sequence for termination of replication (ter) in Escherichia coli and the isolation of the ter-binding protein (TBP) allowed us to test their effects on replication forks initiated at the unique origin of the E. coli chromosome (oriC) in a purified enzyme system. Replication was severely impeded by ter in a unique orientation when purified TBP was supplied to bind it. The target for blockage within the replication complex can now be ascribed to the inability of dnaB helicase to separate the duplex strands when it encounters ter bound by TBP. Other helicases, such as rep and uvrD proteins, that translocate on DNA and displace strands in the direction opposite to that of dnaB protein are also blocked, but only when the TBP-bound ter is oriented in the other direction. From these results, we infer that the orientation of ter confers a particular polarity on the TBP seated on it, such that a helicase is blocked when it confronts TBP from one side, but can act, presumably by displacing TBP, when facing its other side. Thus, the intrinsic nature of the oriented TBP-ter complex is responsible for impeding the helicases, rather than any protein-protein interactions.

In Escherichia coli cells, there is a protein that specifically binds to DNA replication terminus (ter) sites on the host and plasmid genome and then blocks progress of the DNA replication fork. We reported that extract of the cells carrying the plasmid with the tau gene, which was identified to be an essential gene for the termination reaction at the ter site, contained about an 8-fold increase in ter-binding activity of the plasmid-free cells. With improvement of the promoter region of the tau gene on the plasmid by site-directed mutagenesis, the host cells produced the ter-binding protein (Ter protein) over 2,000-fold. Using these over-producing cells as the enzyme source, the Ter protein was purified to apparent homogeneity. Molecular mass 36,000, amino-terminal amino acid sequence (45 residues) and composition of the protein were in good agreement with those deduced from DNA sequence of the tau gene. Footprinting using the purified Ter protein revealed a specific binding to the ter sequences.

Using the "Ter assay" we developed, three separate replication terminus (terC1, terC2, and terC3) sites on the E. coli chromosome were identified. The locations are at 28.3, 35.6, and 33.9 min on the linkage map, respectively. The terC1 site can block the counter-clockwise replication fork only, while the terC2 and terC3 sites inhibit the clockwise fork traveling on the chromosome. DNA sequences of the terC sites required for termination of DNA replication are highly homologous to those of terminus (terR) sites of the R6K plasmid, and the 21 bp consensus DNA sequence of terC is 5'-(A or T) TTAGTTACAACAT (A or C) CT (A or T) (A or T) (A or T) T-3'. In addition, all Ter active pUC-terC plasmids had a low copy number and were unstable in the host cells.

An expression plasmid for calf prochymosin (prorennin) cDNA was constructed. The plasmid (pCR301) contains the lacUV5 promoter in front of the fused gene in which the codons for the N-terminal four amino acids of prochymosin cDNA were replaced with those for the N-terminal ten amino acids of beta-galactosidase. Synthesis of the fused protein with the expected Mr was detected immunologically in Escherichia coli harboring pCR301. The product seemed to be localized in the cell membrane of the bacterial host.