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PubMed2024

Development of SacB-based counterselection for efficient allelic exchange in Fusobacterium nucleatum.

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare, defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knock-in of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possibly related to the natural competence of F. nucleatum. Post-counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research.IMPORTANCEInvestigations into Fusobacterium nucleatum's role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk-based allelic exchange method, although effective, faced an inherent limitation-the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum. Our application of the sacB-based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knock-in at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum.

Fusobacterium nucleatum↗

PubMed2026

Periplasmic SacB as a robust counter-selection tool for genome engineering in the polyploid bacterium Zymomonas mobilis.

UNLABELLED: The alpha-proteobacterium Zymomonas mobilis exhibits exceptional ethanologenic physiology, which makes it a traditional alcoholic beverage producer and a promising chassis for biofuel production. Although genetic tools for this organism have expanded in recent years, a fundamental aspect of its chromosome organization remains to be understood. In particular, Z. mobilis has been suggested to exhibit polyploidy, but this feature is not fully confirmed because of discrepancies among studies reporting the copy number of chromosomes. Here, we tagged the chromosome-partitioning protein ParB with a fluorescent marker to visualize its cellular localization and estimate chromosome copy number in individual cells. Imaging showed that Z. mobilis exhibits several distinctive ParB foci throughout the cytoplasm and an accumulated focus at the pole, indicating that a single Z. mobilis cell contains >5 copies of the chromosome at the oriC regions. We then sought to establish an efficient counter-selection system, which is crucial for engineering multiple copies of the chromosome. We assessed the efficacy of levan-sucrase (SacB) toxicity in Z. mobilis. We found that, despite Z. mobilis secreting a native extracellular sucrase SacB, heterologous periplasmically localized Bacillus subtilis SacB rendered Z. mobilis cells sensitive to sucrose. We successfully used this effect for counter-selection when deleting and inserting targeted DNA sequences into the Z. mobilis genome. Together, this work provides important insights and tools for advancing Z. mobilis genetics and its biotechnological applications. IMPORTANCE: Zymomonas mobilis is a promising industrial bacterium with the capacity to convert sugars into ethanol at nearly maximum theoretical yield. With its expanding use in industrial applications, it is crucial to clarify if individual Z. mobilis cells carry multiple copies of the chromosome, as this has important implications for genome engineering. Two previous studies have used quantitative PCR to address this question, but their reported chromosome copy numbers varied widely from 20 to 100. Here, we used a cell biological approach to estimate the copy number and confirmed that a single Z. mobilis cell possesses multiple copies. In addition, we show that a SacB-based counter-selection works in Z. mobilis, enabling efficient and complete mutation of all chromosome copies.

Zymomonas↗

PubMed1992

Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family.

Induction of the Bacillus subtilis sacB gene and sacPA operon and Escherichia coli bgl operon is mediated by structurally homologous antiterminators encoded by the sacY, sacT, and bglG genes, respectively. When activated, these proteins prevent early transcription termination at terminators located in the leader regions of the three operons. BglG was previously shown to bind in vitro to an imperfectly palindromic 29-nucleotide RNA sequence located upstream of the terminator and partially overlapping with it [Houman, F., Diaz-Torres, M.R. & Wright, A. (1990) Cell 62, 1153-1163]. Similar motifs, here termed ribonucleic antiterminators (RATs), strongly conserved in sequence and in position, are found in the leader of both sacB and sacPA. Mutations were created in sacB RAT and tested in B. subtilis; this showed that sacB RAT is the target for SacY-mediated induction of sacB and that a stem-loop structure in the mRNA is required for regulatory function. Mutations increasing the similarity of the sacB RAT with those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively; most of these changes did not strongly affect induction by SacY, suggesting that the nucleotides at these variable positions act as negative specificity determinants.

Bacillus subtilis↗

PubMed1992

A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.

The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.

Animals↗

PubMed1992

Transcription of the Bacillus subtilis sacX and sacY genes, encoding regulators of sucrose metabolism, is both inducible by sucrose and controlled by the DegS-DegU signalling system.

The adjacent sacX and sacY genes are involved in sucrose induction of the Bacillus subtilis sacB gene by an antitermination mechanism. sacB, encoding the exoenzyme levansucrase, is also subject to regulation by the DegS-DegU signalling system. Using sacXY'-lacZ and sacX'-lacZ fusions, we show that the transcription of the sacX and sacY genes is both inducible by sucrose and regulated by DegU. sacX and sacY appear to constitute an operon, since the deletion of the sacX leader region abolished the expression of a sacXY'-lacZ fusion. The degU-dependent promoter was located by deletion analysis and reverse transcriptase mapping 300 nucleotides upstream from the sacX initiator codon. Sucrose induction of the sacX'-lacZ fusion requires either SacY or the homologous SacT antiterminator, which is involved in sucrose induction of the intracellular sucrase gene (sacPA operon). Sequence analysis of the sacX leader region revealed (20 nucleotides downstream from the transcription start site) a putative binding site for these regulators; however, no structure resembling a rho-independent terminator could be found overlapping this site, unlike the situation for sacPA and sacB. Deletion of a segment of the leader region located 100 nucleotides downstream from this site led to constitutive expression of the sacXY'-lacZ and sacX'-lacZ fusions. These results suggest that the mechanism of sucrose induction of sacXY is different from that of sacPA and sacB.

Bacillus subtilis↗

PubMed1992

Readthrough of the Bacillus subtilis stop codon produces an extended enzyme displaying a higher polymerase activity.

It has been generally accepted that the structural sacB gene of Bacillus subtilis levansucrase encodes a 50,000 Da extracellular protein. However, examination of the DNA sequence of the sacB flanking regions shows a putative open reading frame coding for a 20 amino acid peptide downstream immediately following the terminal TAA stop codon. By site-directed mutagenesis we have changed this stop codon to a glutamine codon. This stop codon readthrough leads to the synthesis and secretion by B. subtilis of a levansucrase possessing an extended polypeptide chain. The extended levansucrase has a molecular weight of 53,000 with a new carboxyl-terminus, rich in basic and hydrophobic amino acids and possessing one cysteine residue. This enzyme synthesizes fructosyl polymer levan of higher molecular weight than the shorter levansucrase. The increase in molecular weight was achieved by increasing the number of branches. These results suggest that the C-terminal part of the enzyme plays a specific role in the degree of branching of the synthesized polymer. Moreover, the extended enzyme is able to form an active dimer from two polypeptide chains linked by an S-S bridge.

Amino Acid Sequence↗

PubMed2024

New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae.

The emergence of antibiotic-resistant Klebsiella pneumoniae is a significant global health threat that has led to increased morbidity and mortality. This resistance also hinders basic research, as many strains are no longer susceptible to antibiotics commonly used in microbial genetics. Addressing this requires the development of new genetic tools with alternative selective markers. In this report, we introduce new allelic exchange vectors for use in drug-resistant strains. These vectors feature a conditional R6K origin of replication, an origin of transfer, SacB counter-selection, and alternative selectable markers. We validated the vectors by generating unmarked deletions in the K. pneumoniae KPPR1S bla (β-lactamase) and lacZ (β-galactosidase) genes. During this process, we defined optimized conditions for SacB-mediated allelic exchange in KPPR1S, significantly enhancing the efficiency of mutant generation. Furthermore, we demonstrated that lacZ is dispensable for virulence and that the lacZ mutant can serve as a surrogate for wild-type strains in competition assays using the Galleria mellonella infection model. Our findings provide new tools for the efficient genetic manipulation of K. pneumoniae and other drug-resistant bacteria.

Klebsiella pneumoniae↗

PubMed1992

Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity.

The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity.

Bacillus subtilis↗

PubMed2025

Effect of transgene on salt tolerance of tobacco.

To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.

Plants, Genetically Modified↗

PubMed1992

The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase.

Interspecific complementation of a Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.

Amino Acid Sequence↗

PubMed1992

Transcriptional regulation of the Tn5276-located Lactococcus lactis sucrose operon and characterization of the sacA gene encoding sucrose-6-phosphate hydrolase.

The Lactococcus lactis sucrose operon was located on the conjugative transposon Tn5276 and the nucleotide sequence of the sacA gene, encoding sucrose-6-phosphate hydrolase, and its surrounding regions was determined. Northern blot analysis showed that the sucrose operon contains two divergent transcriptional units of 3.2 and 3.6 kb, the expression of which is considerably higher in cells grown on sucrose than in cells grown on glucose. This was confirmed by primer extension studies which demonstrated that transcription is initiated at two sucrose-inducible promoters with a back-to-back organization. The 3.2-kb transcriptional unit includes the sacB gene which most probably encodes the sucrose-specific enzyme II of the phosphotransferase system, and may contain the gene encoding fructokinase. The 3.6-kb transcriptional unit includes genes sacA and sacR. The protein encoded by the sacR gene is likely to be involved in the regulation of the sac operon expression, since its deduced N terminus is homologous to helix-turn-helix DNA-binding domains found in several regulatory proteins.

Amino Acid Sequence↗

PubMed1992

Peptide carrier potentiality of Bacillus subtilis levansucrase.

A synthetic oligodeoxynucleotide encoding the vasopressin peptide was ligated to the 3' terminal codon of sacB, the structural gene of levansucrase. This gene fusion was integrated into the chromosome of a Bacillus subtilis strain able to overproduce levansucrase. The extracellular production of the hybrid protein, consisting of the whole levansucrase primary sequence plus the nine amino acids of the vasopressin peptide added at the C-terminal end, represented 50-55% of that found for the wild-type levansucrase (20 mg l-1). The purified hybrid protein displayed the same conformational stability, protease insensitivity and enzymic properties as the wild-type levansucrase. However, the rate and the yield of the unfolding-folding transition at the pH and temperature used for bacterial growth were lower in the case of the hybrid protein; the latter also required a higher iron concentration to be completely folded.

Bacillus subtilis↗

PubMed1992

Rapid generation of directed and unmarked deletions in Xanthomonas.

We have devised a rapid four-step procedure for the generation of directed and unmarked chromosomal deletions in bacteria, based on the use of a novel cloning vector containing the Bacillus subtilis sacB gene that encodes levansucrase and confers sucrose sensitivity, which can be used for counter-selection. Using this technique, we describe the construction of a 6.5 kb directed and unmarked deletion in a phytopathogenicity region of the chromosome in Xanthomonas campestris. This procedure allows rapid and easy transfer of a wide variety of mutant allelic DNA to the bacterial chromosome, and should be adaptable to various bacteria besides Xanthomonas spp.

Bacillus subtilis↗

PubMed1992

Cloning and characterization of the groESL operon from Bacillus subtilis.

The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

Amino Acid Sequence↗

PubMed1992

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.

Alcaligenes↗