Sonoporation Is an Efficient Tool for Intracellular Fluorescent Dextran Delivery and One-Step Double-Crossover Mutant Construction in Fusobacterium nucleatum

Studies of microorganisms are often hindered by a lack of effective genetic tools. One such example is Fusobacterium nucleatum, a gram-negative anaerobe associated with various human infections, including those causing periodontal disease and preterm birth. The first double-crossover allelic-exchange mutant in F. nucleatum was recently constructed using sonoporation, a novel ultrasound-mediated intracellular delivery method, demonstrating potential for bacterial gene transfection. To better unveil its mechanism, the current study examines the factors affecting the outcome of sonoporation. Delivery of Texas Red-conjugated dextran into F. nucleatum by sonoporation was at least twice as efficient as that by electroporation, and sonoporation was nonbactericidal, unlike electroporation. The delivery efficiency was affected by the acoustic pressure amplitude, the duty cycle, and the quantity of microbubbles used to initiate cavitation but not by the pulse repetition frequency of ultrasound application. To examine the involvement of homologous recombination in sonoporation-mediated mutant construction, the highly conserved recA gene, which carried most of the consensus residues, including the P loop, was identified in F. nucleatum, and a double-crossover recA mutant of F. nucleatum 12230, US1610, was constructed by sonoporation. The mutant exhibited increased sensitivity to UV exposure compared with that of the wild type, indicating that the RecA function in F. nucleatum was conserved. Interestingly, US1610 was also sensitive to ultrasound treatment, suggesting the likely involvement of RecA in postsonoporation repair and survival. Since sonoporation has consistently generated one-step double-crossover mutants in F. nucleatum by use of intact suicide plasmids, this technology may be developed into an efficient tool for streamlining mutant construction in bacteria.     

Identification and characterization of a novel adhesin unique to oral fusobacteria

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.     

Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-deficient mutant.

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.     

recA is required in the induction of pectin lyase and carotovoricin in Erwinia carotovora subsp. carotovora.

Pectin lyase (PNL) and the bacteriocin carotovoricin (CTV) were induced in Erwinia carotovora subsp. carotovora 71 by the DNA-damaging agents mitomycin C, nalidixic acid, and UV light. To determine whether the recA product was involved in the expression of these damage-inducible phenotypes, we cloned the E. carotovora subsp. carotovora recA+ gene, inactivated it by Tn5 insertion, and constructed an E. carotovora subsp. carotovora recA::Tn5 strain by gene replacement via homologous recombination. The RecA- strain was more sensitive to methyl methanesulfonate, nitroquinoline oxide, and UV light than its RecA+ parent. The recA mutation did not affect the production of pectate lyase, polygalacturonase, cellulase, and protease or the ability to cause soft rot of potato tubers. With this mutant, unlike with the RecA+ parent strain, PNL and CTV were not induced by mitomycin C or detected in potato tuber tissue. The RecA+ phenotype, including the inducibility of PNL and CTV, could, however, be restored in the mutant in trans by the recA+ gene from either E. carotovora subsp. carotovora or Escherichia coli. We conclude that, in E. carotovora subsp. carotovora, the recA product is required in the induction of PNL and CTV.     

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.     

Inactivation of the Spirochete recA Gene Results in a Mutant with Low Viability and Irregular Nucleoid Morphology

Recently, we have shown the first evidence for allelic exchange in Leptospira spp. By using the same methodology, the cloned recA of Leptospira biflexa was interrupted by a kanamycin resistance cassette, and the mutated allele was then introduced into the L. biflexa chromosome by homologous recombination. The recA double-crossover mutant showed poor growth in liquid media and was considerably more sensitive to DNA-damaging agents such as mitomycin C and UV light than the wild-type strain. The efficiency of plating of the recA mutant was about 10% of that of the parent strain. In addition, microscopic observation of the L. biflexa recA mutant showed cells that are more elongated than those of the wild-type strain. Fluorescent microscopy of stained cells of the L. biflexa wild-type strain revealed that chromosomal DNA is distributed throughout most of the length of the cell. In contrast, the recA mutant showed aberrant nucleoid morphologies, i.e., DNA is condensed at the midcell. Our data indicate that L. biflexa RecA plays a major role in ensuring cell viability via mechanisms such as DNA repair and, indirectly, active chromosome partitioning.     

The recA gene of Chlamydia trachomatis: Cloning, sequence, and characterization in Escherichia coli

Abstract The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.     

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes.

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.     

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

Construction and characterization of a Streptomyces rimosus recA mutant: the RecA-deficient strain remains viable

Although previously reported attempts to construct recA null mutants in Streptomyces spp. have been unsuccessful, we have used the suicide plasmid pErmdeltaRecA to inactivate the recA gene in Streptomyces rimosus by gene disruption. pErmdeltaRecA carries the erythromycin resistance gene ermE and a 451-bp fragment of the S. rimosus recA gene (encoding amino acids 2-151). An erythromycin-resistant clone with single plasmid integration into the recA gene on the chromosome was analyzed in detail. This clone possesses one inactive copy of recA which lacks the entire promoter region and the ATG start codon, and a second, truncated gene that encodes only first 151 amino acids of the RecA protein. This S. rimiosus rec A mutant can therefore be considered a completely RecA-deficient strain. The mutant strain is highly sensitive to UV light. Introduction of a plasmid carrying the wild type S. rimosus recA gene completely restored the UV resistance of the recA mutant to wild-type levels. recA genes encoding RecA proteins with short deletions at the C-terminus (21 and 51 amino acids) could not fully rescue the UV sensitivity of the S. rimosus recA strain, when introduced in the same way.     

Mutational analysis of the Streptomyces lividans recA gene suggests that only mutants with residual activity remain viable

Temperature-sensitive integration plasmids carrying internal fragments of the Streptomyces lividans TK24 recA gene were constructed and used to inactivate the chromosomal recA gene of S. lividans by gene disruption and gene replacement. Integration of these plasmids resulted in recA mutants expressing C-terminally truncated RecA proteins, as deduced from Southern hybridization experiments. Mutants FRECD2 in which the last 42 amino acids, comprising the variable part of bacterial RecA proteins, had been deleted retained the wild-type phenotype. The S. lividans recA mutant FRECD3 produced a RecA protein lacking 87 amino acids probably including the interfilament contact site. FRECD3 was more sensitive to UV and MMS than the wild-type. Its ability to undergo homologous recombination was impaired, but not completely abolished. Integration of the disruption plasmid pFRECD3 in S. coelicolor“Müller” caused the same mutant phenotype as S. lividans FRECD3. In spite of many attempts no S. lividans recA mutants with deletions of 165 C-terminal amino acids or more were isolated. Furthermore, the recA gene could not be replaced by a kanamycin resistance cassette. These experiments indicate a crucial role of the recA gene in ensuring viability of Streptomyces.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment.

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The phenotype of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans.

RecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans. However, it is still unclear how RecA contributes to the resistance. In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670. The properties of the gene products from the recA mutants were compared. recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670. In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity. RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not. The intracellular LexA level in KI696 was decreased following gamma-irradiation. However, the LexA level in strain rec30 was constant irrespective of irradiation. These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not. While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive. Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D. radiodurans.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Influence of recA mutations on gyrA dependent quinolone resistance.

We examined, in Escherichia coli, the influence of recA mutant alleles on the level of quinolone resistance promoted by mutations in the gyrA gene. We found that the recA142 mutation, abolishing all the activities of RecA protein, greatly reduced the level of resistance to the quinolone ciprofloxacin, whereas the recA430 allele affecting the SOS inducing ability of RecA, reduced ciprofloxacin resistance to a lesser extent. The recA142 mutation did not cause enhancement of ciprofloxacin induced DNA breakage in gyrA mutants, indicating that the stabilization of DNA-gyrase complexes by the quinolone is not influenced by a RecA mutant protein. We suggest that RecA protein plays a role in the repair of quinolone damage, principally through a recombinational mechanism and, to a lesser degree, through the induction of the SOS response.     

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX , which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA . The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se .     

Sonoporation: Mechanistic insights and ongoing challenges for gene transfer

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.