Manganese Privation-Induced Transcriptional Upregulation of the Class IIa Bacteriocin Plantaricin 423 in Lactobacillus plantarum Strain 423.

Plantaricin 423 is produced by Lactobacillus plantarum 423 using the pla biosynthetic operon located on the 8,188-bp plasmid pPLA4. As with many class IIa bacteriocin operons, the pla operon carries biosynthetic genes (plaA, precursor peptide; plaB, immunity; plaC, accessory; and plaD, ABC transporter) but does not carry local regulatory genes. Little is known about the regulatory mechanisms involved in the expression of the apparently regulationless class IIa bacteriocins, such as plantaricin 423. In this study, phylogenetic analysis of class IIa immunity proteins indicated that at least three distinct clades exist, which were then used to subgroup the class IIa operons. It became evident that the absence of classical quorum-sensing genes on mobile bacteriocin-encoding elements is a predisposition of the subgroup that includes plantaricin 423, pediocin AcH/PA-1, divercin V41, enterocin A, leucocin-A and -B, mesentericin Y105, and sakacin G. Further analysis of the subgroup suggested that the regulation of these class IIa operons is linked to transition metal homeostasis in the host. By using a fluorescent promoter-reporter system in Lactobacillus plantarum 423, transcriptional regulation of plantaricin 423 was shown to be upregulated in response to manganese privation. IMPORTANCE Lactic acid bacteria hold huge industrial application and economic value, especially bacteriocinogenic strains, which further aids in the exclusion of specific foodborne pathogens. Since bacteriocinogenic strains are sought after, it is equally important to understand the mechanism of bacteriocin regulation. This is currently an understudied aspect of class IIa operons. Our research suggests the existence of a previously undescribed mode of class IIa bacteriocin regulation, whereby bacteriocin expression is linked to management of the producer's transition metal homeostasis. This delocalized metalloregulatory model may fundamentally affect the selection of culture conditions for bacteriocin expression and change our understanding of class IIa bacteriocin gene transfer dynamics in a given microbiome.

Development of a novel selection/counter-selection system for chromosomal gene integrations and deletions in lactic acid bacteria.

BACKGROUND: The underlying mechanisms by which probiotic lactic acid bacteria (LAB) enhance the health of the consumer have not been fully elucidated. Verification of probiotic modes of action can be achieved by using single- or multiple-gene knockout analyses of bacterial mutants in in vitro or in vivo models. We developed a novel system based on an inducible toxin counter-selection system, allowing for rapid and efficient isolation of LAB integration or deletion mutants. The Lactococcus lactis nisin A inducible promoter was used for expression of the Escherichia coli mazF toxin gene as counter-selectable marker. RESULTS: The flippase (FLP)/flippase recognition target (FRT) recombination system and an antisense RNA transcript were used to create markerless chromosomal gene integrations/deletions in LAB. Expression of NisR and NisK signalling proteins generated stable DNA integrations and deletions. Large sequences could be inserted or deleted in a series of steps, as demonstrated by insertion of the firefly bioluminescence gene and erythromycin resistance marker into the bacteriocin operons or adhesion genes of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. CONCLUSIONS: The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. Although this work focuses on two probiotic LAB strains, L. plantarum 423 and E. mundtii ST4SA, the system developed could be adapted to most, if not all, LAB species.

In vivo bioluminescence imaging of the spatial and temporal colonization of lactobacillus plantarum 423 and enterococcus mundtii ST4SA in the intestinal tract of mice.

BACKGROUND: Lactic acid bacteria (LAB) are major inhabitants and part of the normal microflora of the gastrointestinal tract (GIT) of humans and animals. Despite substantial evidence supporting the beneficial properties of LAB, only a few studies have addressed the migration and colonization of probiotic bacteria in the GIT. The reason for this is mostly due to the limitations, or lack of, efficient reporter systems. Here we describe the development and application of a non-invasive in vivo bioluminescence reporter system to study, in real-time, the spatial and temporal persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the intestinal tract of mice. RESULTS: This study reports on the application of the firefly luciferase gene (ffluc) from Photinus pyralis to develop luciferase-expressing L. plantarum 423 and E. mundtii ST4SA, using a Lactococcus lactis NICE system on a high copy number plasmid (pNZ8048) and strong constitutive lactate dehydrogenase gene promoters (Pldh and STldh). The reporter system was used for in vivo and ex vivo monitoring of both probiotic LAB strains in the GIT of mice after single and multiple oral administrations. Enterococcus mundtii ST4SA reached the large intestine 45 min after gavage, while L. plantarum 423 reached the cecum/colon after 90 min. Both strains predominantly colonized the cecum and colon after five consecutive daily administrations. Enterococcus mundtii ST4SA persisted in faeces at higher numbers and for more days compared to L. plantarum 423. CONCLUSIONS: Our findings demonstrate the efficiency of a high-copy number vector, constitutive promoters and bioluminescence imaging to study the colonization and persistence of L. plantarum 423 and E. mundtii ST4SA in the murine GIT. The system allowed us to differentiate between intestinal transit times of the two strains in the digestive tract. This is the first report of bioluminescence imaging of a luciferase-expressing E. mundtii strain to study colonization dynamics in the murine model. The bioluminescence system developed in this study may be used to study the in vivo colonization dynamics of other probiotic LAB.

Efficacy of Lantibiotic Treatment of Staphylococcus aureus-Induced Skin Infections, Monitored by In Vivo Bioluminescent Imaging.

Staphylococcus aureus is a bacterial pathogen responsible for the majority of skin and soft tissue infections. Antibiotics are losing their efficacy as treatment for skin and soft tissue infections as a result of increased resistance in a variety of pathogens, including S. aureus It is thus imperative to explore alternative antimicrobial treatments to ensure future treatment options for skin and soft tissue infections. A select few lantibiotics, a group of natural defense peptides produced by bacteria, inhibit the growth of numerous clinical S. aureus isolates, including methicillin-resistant strains. In this study, the antimicrobial activities of nisin, clausin, and amyloliquecidin, separately administered, were compared to that of a mupirocin-based ointment, which is commonly used as treatment for S. aureus-induced skin infections. Full-thickness excisional wounds, generated on the dorsal surfaces of mice, were infected with a bioluminescent strain of S. aureus (strain Xen 36). The infections were monitored in real time using in vivo bioluminescent imaging. Lantibiotic treatments significantly reduced the bioluminescence of S. aureus Xen 36 to a level similar to that recorded with mupirocin treatment. Wound closure, however, was more pronounced during lantibiotic treatment. Lantibiotics thus have the potential to be used as an alternative treatment option for S. aureus-induced skin infections.

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice.

Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice.

Proteomic profiling of the acid stress response in Lactobacillus plantarum 423.

Acid tolerance is considered an important characteristic of probiotic bacteria. Lactobacillus plantarum 423 tolerates acidic pH and is the ideal candidate in which to study molecular mechanisms that acid-tolerant lactic acid bacteria employ to survive such conditions. In this study we recorded changes in the protein profile of L. plantarum 423 when exposed to pH 2.5 by using a gel-free nanoLC-MS/MS proteomics approach. In total, 97 proteins were detected as more abundant, and 12 proteins were detected solely when strain 423 was exposed to pH 2.5. General stress response proteins, the utilization of a variety of carbohydrate sources in a glucose rich environment, altered pyruvate metabolism, increased lysine biosynthesis, and a significant oxidative stress response was observed in acid-stressed cells. The accumulation of basic compounds also seemed to play an integral role in the response to acid stress. We observed a marked decrease in proteins involved in cell wall and phospholipid biosynthesis, transcription, translation, and cell division. The most abundant protein detected was an uncharacterized protein, JDM1_2142. Functional analysis revealed that this protein plays a role in survival during acid stress. Our results contribute to the growing body of knowledge on the molecular mechanisms employed by lactobacilli, in particular L. plantarum, to ensure survival in acidic conditions.

Could the COVID-19-Driven Increased Use of Ivermectin Lead to Incidents of Imbalanced Gut Microbiota and Dysbiosis?

The microfilaricidal anthelmintic drug ivermectin (IVM) has been used since 1988 for treatment of parasitic infections in animals and humans. The discovery of IVM's ability to inactivate the eukaryotic importin α/ß1 heterodimer (IMPα/ß1), used by some viruses to enter the nucleus of susceptible hosts, led to the suggestion of using the drug to combat SARS-CoV-2 infection. Since IVM has antibacterial properties, prolonged use may affect commensal gut microbiota. In this review, we investigate the antimicrobial properties of IVM, possible mode of activity, and the concern that treatment of individuals diagnosed with COVID-19 may lead to dysbiosis.

Molecular insights into probiotic mechanisms of action employed against intestinal pathogenic bacteria.

Gastrointestinal (GI) diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species that have developed resistance to antibiotics. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Several studies have documented a reduction, or prevention, of GI diseases by probiotic bacteria. Since the activities of probiotic bacteria are closely linked with conditions in the host's GI-tract (GIT) and changes in the population of enteric microorganisms, a deeper understanding of gut-microbial interactions is required in the selection of the most suitable probiotic. This necessitates a deeper understanding of the molecular capabilities of probiotic bacteria. In this review, we explore how probiotic microorganisms interact with enteric pathogens in the GIT. The significance of probiotic colonization and persistence in the GIT is also addressed.

Survival of Planktonic and Sessile Cells of Lactobacillus rhamnosus and Lactobacillus reuteri upon Exposure to Simulated Fasting-State Gastrointestinal Conditions.

In this study, we report on the formation and resilience of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 biofilms cultivated in a CO2 evolution measurement system (CEMS) and exposed to biologically relevant, fasting-state gastrointestinal fluids under continuous flow conditions. For comparative purposes, planktonic and sessile populations of L. reuteri HFI-LD5 and L. rhamnosus HFI-K2 were each exposed to fasting-state gastric fluid (FSGF, pH 2.0) for 2 h, fasting-state intestinal fluid (FSIF, pH 7.5) for 6 h, and simulated colonic fluid (SCoF, pH 7.0) for 24 h. Planktonic cell numbers of L. reuteri HFI-LD5 declined from 6.6 log10 CFU/mL to 3.2 log10 CFU/mL and L. rhamnosus HFI-K2 from 6.6 log10 CFU/mL to undetectable levels after exposure to FSGF. Limited loss in viability was observed when free-floating cells were exposed to FSIF and SCoF. Sessile populations of both strains survived and recovered from the sequential exposure to all three gastric fluids despite observed detachment of biofilm biomass and a temporary decrease in metabolic activity to below detection limits, as recorded by changes in whole-biofilm CO2 production rates. The planktonic cell-focused gut microbiome-related research has most likely caused an underestimation in the overall survival ability of microorganisms in the gastrointestinal tract. Sessile cells of L. reuteri HFI-LD5 were metabolically inactive when exposed to gastric (FSGF) and intestinal (FSIF) fluids, suggesting that biofilms are formed in the small intestinal tract as survival mechanism. In the case of L. rhamnosus HFI-K2, cells were released from biofilms when suddenly exposed to pH 2.0.

The Effect of Vancomycin on the Viability and Osteogenic Potential of Bone-Derived Mesenchymal Stem Cells.

Traditionally, methicillin-resistant Staphylococcus aureus (MRSA) is treated with vancomycin, administrated intravenously or applied directly onto infected tissue. The effect of direct (as opposed to systemic) vancomycin treatment on bone formation and remodelling is largely unknown. The minimal inhibitory concentration (MIC) of vancomycin was determined by adding 200 µL of different concentrations (1-20 µg/mL) to actively growing cultures of S. aureus Xen 31 (methicillin-resistant) and S. aureus Xen 36 (methicillin-sensitive), respectively, and recording changes in optical density over 24 h. Bone marrow-derived and proximal femur-derived mesenchymal stem cells (bmMSCs and pfMSCs) from rat femora were exposed to 1 × MIC (5 µg/mL) and 4 × MIC (20 µg/mL) of vancomycin for 7 days. Cell viability was determined by staining with crystal violet and MTT (3-(4,5- di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), respectively, and osteogenic differentiation by staining with Alizarin Red S. Vancomycin had no effect on the viability of bmMSCs and pfMSCs, even at high levels (20 µg/mL). The osteogenic differentiation of pfMSCs was partially inhibited, while osteogenesis in bmMSCs was not severely affected. The direct application of vancomycin to infected bone tissue, even at excessive levels, may preserve the viability of resident MSC populations.

Antibacterial Activity of Vancomycin Encapsulated in Poly(DL-lactide-co-glycolide) Nanoparticles Using Electrospraying.

Vancomycin is often used to treat infections caused by ß-lactam-resistant bacteria. However, methicillin-resistant strains of Staphylococcus aureus (MRSA) acquired resistance to vancomycin, rendering it less effective in the treatment of serious infections. In the search for novel antibiotics, alternative delivery mechanisms have also been explored. In this study, we report on the encapsulation of vancomycin in PLGA [poly(DL-lactide-co-glycolide)] nanoparticles by electrospraying. The nanoparticles were on average 247 nm in size with small bead formations on the surface. Clusters of various sizes were visible under the SEM (scanning electron microscope). Vancomycin encapsulated in PLGA (VNP) was more effective in inhibiting the growth of S. aureus Xen 31 (MRSA) and S. aureus Xen 36 than un-encapsulated vancomycin. Encapsulated vancomycin had a minimum inhibitory concentration (MIC) of 1 µg/mL against MRSA compared to 5 µg/mL of free vancomycin. At least 70% (w/w) of the vancomycin was encapsulated. Thirty percent of the vancomycin was released within the first 144 h, followed by slow release over 10 days. Vancomycin encapsulated in PLGA nanoparticles may be used to treat serious infections.

Migration of Bacteriocins Across Gastrointestinal Epithelial and Vascular Endothelial Cells, as Determined Using In Vitro Simulations.

Little is known about the migration of bacteriocins across human cells. In this study, we report on migration of three bacteriocins nisin, plantaricin 423 and bacST4SA across colonic adenocarcinoma (Caco-2) cells and human umbilical vein endothelial cells (HUVECs). Bacteriocins were fluorescently labelled while still maintaining antimicrobial activity. Migration of fluorescently labelled bacteriocins across monolayers was assessed in vitro using transmigration well inserts. After 3 h, 75% of nisin, 85% of plantaricin 423 and 82% of bacST4SA migrated across the Caco-2 cell monolayer. Over the same time span, 88% nisin, 93% plantaricin 423 and 91% bacST4SA migrated across the HUVEC monolayer. The viability of both cell types remained unchanged when exposed to 50 µM of nisin, plantaricin 423 or bacST4SA. The effect of human plasma on bacteriocin activity was also assessed. Activity loss was dependent on bacteriocin type and concentration, with the class-IIa bacteriocins retaining more activity compared to nisin. This is the first report of bacteriocins migrating across simulated gastrointestinal- and vascular-barriers. This study provides some of the first evidence that bacteriocins are capable of crossing the gut-blood-barrier. However, in vivo studies need to be performed to confirm these findings and expand on the role of bacteriocin migration across cell barriers.

Presence of a family of plasmids (29 to 65 kilobases) with a 26-kilobase common region in different strains of the sulfur-oxidizing bacterium Acidithiobacillus caldus.

Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.

Aciduric Strains of Lactobacillus reuteri and Lactobacillus rhamnosus, Isolated from Human Feces, Have Strong Adhesion and Aggregation Properties.

Human feces were streaked onto MRS Agar adjusted to pH 2.5, 3.0, and 6.4, respectively, and medium supplemented with 1.0% (w/v) bile salts. Two aciduric strains, identified as Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 (based on 16S rDNA and recA sequences), were non-hemolytic and did not hydrolyze mucin. The surface of Lactobacillus reuteri HFI-LD5 cells has a weak negative charge, whereas Lactobacillus rhamnosus HFI-K2 has acidic and basic properties, and produces exopolysaccharides (EPS). None of the strains produce bacteriocins. Both strains are resistant to several antibiotics, including sulfamethoxazole-trimethoprim and sulphonamides. The ability of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 to grow at pH 2.5 suggests that they will survive passage through the stomach. EPS production may assist in binding to intestinal mucus, especially in the small intestinal tract, protect epithelial cells, and stimulate the immune system. Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 may be used as probiotics, especially in the treatment of small intestinal bacterial overgrowth (SIBO).

Reporter systems for in vivo tracking of lactic acid bacteria in animal model studies.

Bioluminescence (BLI) and fluorescence imaging (FI) allow for non-invasive detection of viable microorganisms from within living tissue and are thus ideally suited for in vivo probiotic studies. Highly sensitive optical imaging techniques detect signals from the excitation of fluorescent proteins, or luciferase-catalyzed oxidation reactions. The excellent relation between microbial numbers and photon emission allow for quantification of tagged bacteria in vivo with extreme accuracy. More information is gained over a shorter period compared to traditional pre-clinical animal studies. The review summarizes the latest advances in in vivo bioluminescence and fluorescence imaging and points out the advantages and limitations of different techniques. The practical application of BLI and FI in the tracking of lactic acid bacteria in animal models is addressed.

Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank.

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.

Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs.

All strains of the moderately thermophilic, acidophilic, sulphur-oxidizing bacterium Acidithiobacillus caldus that have been tested contain a set of chromosomal arsenic resistance genes. Highly arsenic-resistant strains isolated from commercial arsenopyrite bio-oxidation tanks contain additional transposon-located (TnAtcArs) arsenic resistance genes. The chromosomal At. caldus ars genes were cloned and found to consist of arsR and arsC genes transcribed in one direction, and arsB in the opposite direction. The arsRC genes were co-transcribed with ORF1, and arsB with ORF5 in both At. caldus and Escherichia coli, although deletion of ORFs 1 and 5 did not appear to affect resistance to arsenate or arsenite in E. coli. ORFs 1 and 5 have not previously been reported as part of the ars operons, and had high amino acid identity to hypothetical proteins from Polaromonas naphthalenivorus (76%) and Legionella pneumophila (60%), respectively. Reporter-gene studies showed that the arsenic operon of transposon origin (TnAtcArs) was expressed at a higher level, and was less tightly regulated in E. coli than were the At. caldus ars genes of chromosomal origin. Plasmid pSa-mediated conjugal transfer of TnAtcArs from E. coli to At. caldus strains lacking the transposon was successful, and resulted in greatly increased levels of resistance to arsenite.

An unusual Tn21-like transposon containing an ars operon is present in highly arsenic-resistant strains of the biomining bacterium Acidithiobacillus caldus.

A transposon, TnAtcArs, that carries a set of arsenic-resistance genes was isolated from a strain of the moderately thermophilic, sulfur-oxidizing, biomining bacterium Acidithiobacillus caldus. This strain originated from a commercial plant used for the bio-oxidation of gold-bearing arsenopyrite concentrates. Continuous selection for arsenic resistance over many years had made the bacterium resistant to high concentrations of arsenic. Sequence analysis indicated that TnAtcArs is 12 444 bp in length and has 40 bp terminal inverted repeat sequences and divergently transcribed resolvase and transposase genes that are related to the Tn21-transposon subfamily. A series of genes consisting of arsR, two tandem copies of arsA and arsD, two ORFs (7 and 8) and arsB is situated between the resolvase and transposase genes. Although some commercial strains of At. caldus contained the arsDA duplication, when transformed into Escherichia coli, the arsDA duplication was unstable and was frequently lost during cultivation or if a plasmid containing TnAtcArs was conjugated into a recipient strain. TnAtcArs conferred resistance to arsenite and arsenate upon E. coli cells. Deletion of one copy of arsDA had no noticeable effect on resistance to arsenite or arsenate in E. coli. ORFs 7 and 8 had clear sequence similarity to an NADH oxidase and a CBS-domain-containing protein, respectively, but their deletion did not affect resistance to arsenite or arsenate in E. coli. TnAtcArs was actively transposed in E. coli, but no increase in transposition frequency in the presence of arsenic was detected. Northern hybridization and reporter gene studies indicated that although ArsR regulated the 10 kb operon containing the arsenic-resistance genes in response to arsenic, ArsR had no effect on the regulation of genes associated with transposition activity.

Plasmid evolution and interaction between the plasmid addiction stability systems of two related broad-host-range IncQ-like plasmids.

Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmids are likely to encounter each other. We investigated the relative efficiencies of the two stability systems and whether they had evolved apart sufficiently for each pas to stabilize a plasmid in the presence of the other. The three-component pTF-FC2 pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells (+/- 92% and +/- 60% after 100 generations, respectively). The PasA antidote of each pas was unable to neutralize the PasB toxin of the other plasmid. The pas proteins of each plasmid autoregulated their own expression as well as that of the pas of the other plasmid. The pas of pTF-FC2 was more effective at repressing the pas operon of pTC-F14 than the pas of pTC-F14 was able to repress itself or the pas of pTF-FC2. This increased efficiency was not due to the PasC of pTF-FC2. The effect of this stronger repression was that pTF-FC2 displaced pTC-F14 when the two plasmids were coresident in the same E. coli host cell. Plasmid curing resulted in the arrest of cell growth but did not cause cell death, and plasmid stability was not influenced by the E. coli mazEF genes.