A small cryptic plasmid from Rhodococcus erythropolis: characterization and utility for gene expression.

Exploration of metabolically diverse rhodococci is generally hampered by the lack of genetic tools. A small cryptic plasmid (pAN12) isolated from Rhodococcus erythropolis strain AN12 was sequenced. Plasmid pAN12 encodes proteins that share homology to replication proteins and putative cell division proteins. Based on in vitro transposon mutagenesis, we determined that the Rep protein of pAN12 is essential for plasmid replication in Rhodococcus spp., and the putative cell division protein Div is important for plasmid stability. The pAN12 replicon is able to replicate in R. erythropolis strains AN12 and CW23 (ATCC 47072) and is compatible with the nocardiophage Q4 replicon present on a Rhodococcus shuttle plasmid pDA71. pAN12 appears to belong to the pIJ101/pJV1 family of rolling circle replication plasmids. Expression of an isoprenoid pathway gene ( dxs) on the pAN12-derived multicopy shuttle vector increased production of carotenoid pigments in R. erythropolis ATCC 47072.

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Skewed oligomers and origins of replication.

The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin. The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H. pylori, M. thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown. In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions. An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin. Possible explanations for the phenomenon of skewed oligomers are discussed. Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.

A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake.

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM, is induced during competence development in wild-type H. influenzae but not in RJ248.

In vitro Tn7 mutagenesis of Haemophilus influenzae Rd and characterization of the role of atpA in transformation.

Haemophilus influenzae Rd is a gram-negative bacterium capable of natural DNA transformation. The competent state occurs naturally in late exponential growth or can be induced by a nutritional downshift or by transient anaerobiosis. The genes cya, crp, topA, and sxy (tfoX) are known to function in the regulation of competence development. The phosphoenolpyruvate:carbohydrate phosphotransferase system functions to maintain levels of cyclic AMP necessary for competence development but is not directly involved in regulation. The exact signal(s) for competence and the genes that mediate the signal(s) are still unknown. In an effort to find additional regulatory genes, H. influenzae Rd was mutated by using an in vitro Tn7 system and screened for mutants with a reduced ability to induce the competence-regulatory gene, comA. Insertions in atpA, a gene coding for the alpha subunit of the F1 cytoplasmic domain of the ATP synthase, reduce transformation frequencies about 20-fold and cause a significant reduction in expression of competence-regulatory genes, while the expression of constitutive competence genes is only minimally affected. In addition, we found that an insertion in atpB, which encodes the a subunit of the F0 membrane-spanning domain, has a similar effect on transformation frequencies.

Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.

The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

The complete genome sequence of the gastric pathogen Helicobacter pylori.

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

Role of the two-component signal transduction and the phosphoenolpyruvate: carbohydrate phosphotransferase systems in competence development of Haemophilus influenzae Rd.

Haemophilus influenzae Rd becomes competent for transformation by nutritional downshift or transient anaerobic growth through a process that requires cyclic AMP receptor protein and adenylate cyclase. Insertion mutations in crr or ptsI of the phosphoenolpyruvate:carbohydrate phosphotransferase system lowered transformation frequencies, and the effect was reversed by the addition of cyclic AMP. However, insertions into H. influenzae homologs of two-component signal transduction genes had no effect on competence.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenzae Rd genome.

The naturally transformable, Gram-negative bacterium Haemophilus influenzae Rd preferentially takes up DNA of its own species by recognizing a 9-base pair sequence, 5'-AAGTGCGGT, carried in multiple copies in its chromosome. With the availability of the complete genome sequence, 1465 copies of the 9-base pair uptake site have been identified. Alignment of these sites unexpectedly reveals an extended consensus region of 29 base pairs containing the core 9-base pair region and two downstream 6-base pair A/T-rich regions, each spaced about one helix turn apart. Seventeen percent of the sites are in inverted repeat pairs, many of which are located downstream to gene termini and are capable of forming stem-loop structures in messenger RNA that might function as signals for transcription termination.

A periplasmic protein disulfide oxidoreductase is required for transformation of Haemophilus influenzae Rd.

The mutated gene in JG16, a Haemophilus influenzae strain deficient in competence-induced DNA binding and uptake, was cloned and the wild-type allele was sequenced. The gene was shown by Northern analysis to be constitutively expressed on a 1.7-kilobase transcript. The gene product was identified as a 20.6-kDa protein targeted to the periplasm. The protein contains the sequence Cys-Pro-His-Cys (CPHC) and is highly similar to two other periplasmic CPHC motif-containing proteins: DsbA, an Escherichia coli protein (45% identity, 87% homology) and TcpG, a Vibrio cholerae protein (32% identity, 74% homology). Both DsbA and TcpG promote disulfide bond formation in periplasmic proteins, are required for pilus biogenesis, and, like thioredoxin, are capable of reducing insulin in vitro. The Haemophilus protein was shown to complement an E. coli mutation in DsbA and was named Por (periplasmic oxidoreductase). In JG16 the competence-dependent redistribution of inner membrane proteins did not occur. These findings suggest that Por is required for the correct assembly and/or folding of one or more disulfide-containing cell envelope protein involved either in competence development or in the DNA-binding and -uptake machinery.

Nucleotide sequence of a cluster of genes involved in the transformation of Haemophilus influenzae Rd.

A genetic locus implicated in the development of competence in Haemophilus influenzae Rd has been previously mapped to a 12.8-kb PstI region of the chromosome [Tomb et al., J. Bacteriol. 171 (1989) 3796-3802]. To define the boundaries of this locus and to identify the gene(s) involved in transformation, additional mini-Tn10kan mutagenesis was performed and the region containing all mutagenic insertions was sequenced. Three new transformation-deficient (Tfo-) mutants were found, bringing the number of distinct mutations mapped to this region up to eight. The transformation frequency of strains carrying the new insertions was 25- to 10(5)-fold less than wild type. The ends of the mini-Tn10kan element were used as starting points to sequence a 9.1-kb region. The position of the eight mutagenic insertions was determined and ten putative open reading frames (ORFs) were found. One of the mini-Tn10kan elements had inserted in an intergenic region while the rest had inserted in six of the ORFs. Based on the phenotypes of the mutant strains and the position of the insertions, we concluded that at least three of the genes should be involved in transformation. In addition, fourteen 9-11-bp uptake signal sequences (USS) were found, four of which were part of stem-loop structures and could function as attenuators of terminators of transcription.

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.

Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.