Mars Extant Life: What's Next? Conference Report.

On November 5-8, 2019, the "Mars Extant Life: What's Next?" conference was convened in Carlsbad, New Mexico. The conference gathered a community of actively publishing experts in disciplines related to habitability and astrobiology. Primary conclusions are as follows: A significant subset of conference attendees concluded that there is a realistic possibility that Mars hosts indigenous microbial life. A powerful theme that permeated the conference is that the key to the search for martian extant life lies in identifying and exploring refugia ("oases"), where conditions are either permanently or episodically significantly more hospitable than average. Based on our existing knowledge of Mars, conference participants highlighted four potential martian refugium (not listed in priority order): Caves, Deep Subsurface, Ices, and Salts. The conference group did not attempt to reach a consensus prioritization of these candidate environments, but instead felt that a defensible prioritization would require a future competitive process. Within the context of these candidate environments, we identified a variety of geological search strategies that could narrow the search space. Additionally, we summarized a number of measurement techniques that could be used to detect evidence of extant life (if present). Again, it was not within the scope of the conference to prioritize these measurement techniques-that is best left for the competitive process. We specifically note that the number and sensitivity of detection methods that could be implemented if samples were returned to Earth greatly exceed the methodologies that could be used at Mars. Finally, important lessons to guide extant life search processes can be derived both from experiments carried out in terrestrial laboratories and analog field sites and from theoretical modeling.

Complete Genome Sequence and Methylome Analysis of Micrococcus luteus SA211, a Halophilic, Lithium-Tolerant Actinobacterium from Argentina.

Micrococcus luteus has been found in a wide range of habitats. We report the complete genome sequence and methylome analysis of strain SA211 isolated from a hypersaline, lithium-rich, high-altitude salt flat in Argentina with single-molecule real-time sequencing.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development.

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'-region, named sopB4 and sopB5, were each fused to the gvpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence.

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.

Antigen presentation using novel particulate organelles from halophilic archaea.

A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea. Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology. When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic. Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant. Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory. Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.

Genomic and genetic dissection of an archaeal regulon.

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix-turn-helix DNA binding motif. We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a gamma-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine-pyrimidine sequences (RY boxes) near the regulated promoters.

brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon.

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176-12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology.

Genome sequence of Halobacterium species NRC-1.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity.

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3' to the TATA box within an 11 bp alternating purine-pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5' to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3' end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker.

To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Deltaura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-5'-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Deltabop strain was constructed by transforming a Deltaura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Deltabop recombinants at a frequency of > 10-2 in both approaches. These studies establish an efficient new genetic strategy towards the systematic knockout of genes in an archaeon.

Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?

Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons. Previously, the analysis of one such replicon, pNRC100, in Halobacterium sp. strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs). Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families. A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs. More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases. Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids. Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100. However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found. A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes. The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

Isolation and chromosomal distribution of natural Z-DNA-forming sequences in Halobacterium halobium.

Conditions favoring left-handed Z-DNA such as high salinity (> 4 ), high negative DNA supercoiling, and GC-rich DNA [statistically favoring d(CG)n repeat sequences], are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium. In order to identify and study Z-DNA regions of the H. halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed. Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient. In control experiments using mixtures of pUC12 (Z-negative) and a d(CG)5-containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure. The selection efficiency was determined by transformation of Escherichia coli DH5alpha with eluted plasmids and blue-white screening on X-gal plates. Twenty recombinant plasmids containing Z-DNA-forming sequences of H. halobium were isolated from a genomic library using affinity chromatography. Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody. Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids. The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H. halobium Z-DNA segments as probe. Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.

Analysis of left-handed Z-DNA formation in short d(CG)n sequences in Escherichia coli and Halobacterium halobium plasmids. Stabilization by increasing repeat length and DNA supercoiling but not salinity.

To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)n sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed. Z-DNA conformation in the d(CG)n sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay. Using the ZIBS assay on plasmids purified from E. coli, the transition from B-DNA to Z-DNA occurred from d(CG)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation. These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences. Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations. When a plasmid containing d(CG)4, was isolated from a topA mutant of E. coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region. In plasmids purified from H. halobium, which showed 30% greater linking deficiency than from E. coli, 20-40% greater Z-DNA formation was found in d(CG)4-6 sequences. Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E. coli or H. halobium in the NaCl concentration range of 0.1-4 M.

Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure.

The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site approximately 480 bp upstream of bop showed that a 53-bp region 5' to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5' to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO4, which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.

Wild-type gas vesicle formation requires at least ten genes in the gvp gene cluster of Halobacterium halobium plasmid pNRC100.

To study the functions of the 13 gvp genes, gvpMLKJIHGFEDACN, on plasmid pNRC100 of Halobacterium halobium in gas vesicle formation, we carried out linker scanning mutagenesis of the gene cluster. We constructed a 24.5-kb Escherichia coli-H. halobium shuttle plasmid, pFL2, containing the gvp gene cluster and introduced a kanamycin resistance (kappa) cassette into each gene (except for gvpA). Transformation of H. halobium SD109, which had the entire gvp gene cluster deleted, with pFL2 and mutated pFL2 derivatives showed that while the unmutated gene cluster successfully programmed gas vesicle formation, derivatives with insertion of the kappa cassette in any of the gvp genes, except gvpM, did not lead to production of normal gas vesicles. Insertions in gvpL, -K, -J, -I, and -F resulted in a complete block in gas vesicle synthesis, while insertions in gvpH, -G, -E, -D, -C, and -N resulted in greatly reduced gas vesicle synthesis. In most cases, the block in gas vesicle synthesis did not result from polar effects, since similar results were obtained for derivatives of the insertion mutants in which most of the internal portion of the kappa cassette was deleted and only small (15 to 54-bp) insertions remained. The only exceptions were for gvpH and gvpD, where deletion of the internal portion of the kappa insertions resulted in phenotypic reversion. Electron microscopic analysis of the kappa mutants revealed that interruptions of gvpC and gvpN result in the formation of smaller gas vesicle than in the wild type, while interruptions of gvpF, -G, -H, -J, -K, and -L produce no discernible vesicle intermediates. These results indicate the gvpA, -C, and -N, which have the rightward transcriptional orientation, encode structural proteins, with gvpC and gvpN necessary for late stages of vesicle formation, and gvpL, -K, -J, -I, -H, -G, and -F, which have the leftward transcriptional orientation encode proteins involved in early steps in the assembly of gas vesicles.

Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100.

We have identified the replication origin of pNRC100, a 200-kb plasmid of Halobacterium halobium, by assaying for replication ability of miniplasmids containing cloned fragments of pNRC100 and the mevinolin resistance selectable marker of Haloferax volcanii. First, we showed the replication ability of plasmid pNGHCMEV1, which contains the 19-kb HindIII-C fragment of pNRC100, by recovery of plasmid DNA from mevinolin-resistant transformants of H. halobium. The minimal replication origin of approximately 3.9 kb was defined by subcloning successively smaller regions of pNGHCMEV1 and assaying for plasmid replication in either H. halobium or H. volcanii. The same replication origin was also recovered after transformation of H. volcanii with a library of partial Sau3AI fragments of pNRC100. The nucleotide sequence of the minimal replication origin was determined and found to contain a long open reading frame, named repH, transcribed away from a highly A+T-rich region. The transcription start site was identified by primer extension analysis to be 17 to 18 nucleotides 5' to a putative repH start codon. The predicted product of the repH gene, an acidic protein with a molecular weight of 113,442, showed 24 to 27% identity with predicted gene products of H. volcanii plasmid pHV2 and H. halobium plasmid p phi HL, suggesting that each is involved in plasmid replication. One pNRC100 minireplicon, pNG11 delta 12, was analyzed by linker scanning mutagenesis, which showed the requirement of repH for replication. Restoration of the repH reading frame of one replication-defective pNG11 delta 12 derivative by introduction of a second small insertion resulted in reversion to replication proficiency. The replication ability of pNG11delta12 was lost when the entire A+T-rich region, about 550 bp long, was deleted but not when small insertions or deletions were introduced into this region. The presence of only 52 bp of the A+T-rich segment was sufficient to permit replication. The pNG11delta12 minireplicon was lost at high frequency from cells grown without mevinolin selection, suggesting that the plasmid partitioning locus of pNRC100 is absent in the minimal replication origin region. We discuss the possible roles of the repH gene and the A+T-rich region in replication of pNRC100.