A third genetic locus required for the formation of heterocysts in Anabaena sp. strain PCC 7120.

Mutagenesis of Anabaena sp. strain PCC 7120 with a derivative of transposon Tn5 led to the isolation of a mutant strain, P6, in which heterocysts are not formed (A. Ernst, T. Black, Y. Cai, J.-M. Panoff, D. N. Tiwari, and C. P. Wolk, J. Bacteriol. 174:6025-6032, 1992). Reconstruction of the transposon mutation of P6 in the wild-type strain reproduced the phenotype of the original mutant. Analysis by pulsed-field gel electrophoresis localized the transposition at ca. 3.44 Mb on the physical map of the chromosome of wild-type Anabaena sp. The transposon was situated within an open reading frame (ORF), which we denote hetP, whose wild-type form was cloned and also sequenced. The predicted HetP protein was not found to show significant sequence similarity to other proteins. The mutation in strain P6 could be complemented by a clone of a fragment of wild-type DNA that includes hetP and at least one additional ORF 3' from hetP, but not by a clone that includes hetP as its only ORF. The latter clone proved highly toxic. The phenotype of the P6 mutant may, therefore, be due to a polar effect of the insertion of the transposon. Filaments of strain P6 and of the wild-type strain, when bearing the complementing fragment on a pDU1-based plasmid, showed an increased frequency of clustered heterocysts compared with that of the wild-type strain.     

Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.     

Unusual organization associated to a tandem of IS231 may yield two peculiar cloverleaf secondary structures.

A 5.7-kb EcoRI fragment was cloned from plasmid DNA of Bacillus thuringiensis strain M15. It contains two insertion sequences (IS), IS231M2 and -M1 in the 5'-3' order, arranged in tandem, in same orientation, separated by a 540-bp region. The primary structure is typical of a composite transposon, here of 3847 bp in length, for which the name Tn231M is proposed. Each IS is delimited by 18-bp inverted repeats (IR), and flanked by 11-bp direct repeats (DR). Both IS share 99.3% nucleotide identities. IS231M1 has a single open reading frame (ORF) which encodes a putative 477-amino-acid transposase. IS231M2 has two smaller ORFs: ORF1 and ORF2, which could code for polypeptides of 329 and 118 amino acids in length, respectively. Further analysis reveals that the regions upstream of IS231M2, and downstream of -M1, and the 540-bp region, contain additional pairs of IR and DR. Interestingly, potential annealing between all pairs of IR and DR could generate two unusual cloverleaf secondary structures.     

Evidence that the hanA gene coding for HU protein is essential for heterocyst differentiation in, and cyanophage A-4(L) sensitivity of, Anabaena sp. strain PCC 7120.

The highly pleiotropic, transposon-generated mutant AB22 of Anabaena sp. strain PCC 7120 exhibits slow growth, altered pigmentation, cellular fragility, resistance to phage A-4(L), and the inability to differentiate heterocysts. Reconstruction of the transposon mutation in the wild-type strain reproduced the phenotype of the original mutant. Sequencing of the flanking DNA showed that the transposon had inserted at the beginning of a gene, which we call hanA, that encodes Anabaena HU protein (R. Nagaraja and R. Haselkorn, Biochimie 76:1082-1089, 1994). Mapping of the transposon insertion by pulsed-field gel electrophoresis showed that hanA is located at ca. 4.76 Mb on the physical map of the chromosome and is transcribed clockwise. Repeated subculturing of AB22 resulted in improved growth and loss of filament fragmentation, presumably because of one or more compensatory mutations; however, the mutant retained its A-4(L)r Het- phenotype. The mutation in strain AB22 could be complemented by a fragment of wild-type DNA bearing hanA as its only open reading frame.     

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.     

Characterization of transposon Tn5469 from the cyanobacterium Fremyella diplosiphon.

A transposon, designated Tn5469, was isolated from mutant strain FdR1 of the filamentous cyanobacterium Fremyella diplosiphon following its insertion into the rcaC gene. Tn5469 is a 4,904-bp noncomposite transposon with 25-bp near-perfect terminal inverted repeats and has three tandemly arranged, slightly overlapping potential open reading frames (ORFs) encoding proteins of 104.6 kDa (909 residues), 42.5 kDa (375 residues), and 31.9 kDa (272 residues). Insertion of Tn5469 into the rcaC gene in strain FdR1 generated a duplicate 5-bp target sequence. On the basis of amino acid sequence identifies, the largest ORF, designated tnpA, is predicted to encode a composite transposase protein. A 230-residue domain near the amino terminus of the TnpA protein has 15.4% amino acid sequence identity with a corresponding domain for the putative transposase encoded by Lactococcus lactis insertion sequence S1 (ISS1). In addition, the sequence for the carboxyl-terminal 600 residues of the TnpA protein is 20.0% identical to that for the TniA transposase encoded by Tn5090 on Klebsiella aerogenes plasmid R751. The TnpA and TniA proteins contain the D,D(35)E motif characteristic of a recently defined superfamily consisting of bacterial transposases and integrase proteins of eukaryotic retroelements and retrotransposons. The two remaining ORFs on Tn5469 encode proteins of unknown function. Southern blot analysis showed that wild-type F. diplosiphon harbors five genomic copies of Tn5469. In comparison, mutant strain FdR1 harbors an extra genomic copy of Tn5469 which was localized to the inactivated rcaC gene. Among five morphologically distinct cyanobacterial strains examined, none was found to contain genomic sequences homologous to Tn5469.     

Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.     

中华根瘤菌自体诱导物合成酶基因的筛选及其在大肠杆菌中的表达

通过携带有mariner转座子的质粒pJZ290随机插入诱变中华根瘤菌(Sinorhizobium meliloti)建立突变子文库,并从中筛选到自体诱导物(autoinducer,AI)部分缺失突变株YW1。Arbitrary PCR扩增、DNA测序得到YW1基因组DNA中mariner转座子两端侧翼序列,经DNA序列拼接在GenBank上进行同源性分析后获得一个621bp的完整的开放阅读框(ORF),该ORF编码的酶具有206个氨基酸,与草木樨中华根瘤菌(Sinorhizobium medicae)WSM419的LuxI类自体诱导物合成酶(autoinducer synthase)TraI的同源性高达99%。因此,也将该基因命名为traⅠ。将该基因克隆到广宿主范围表达载体pYC12并在大肠杆菌Escherichia coli DH5α中成功表达,C18反相薄层层析(TLC)在阳性重组子培养上清中检测到四种自体诱导物分子,其中的两种正是AI缺失突变株YW1所缺失的AI,这些结果表明该traⅠ基因在苜蓿中华根瘤菌负责合成两种自体诱导物分子,为进一步研究其群体感应系统奠定了理论基础。     

Importance of opgHXcv of Xanthomonas campestris pv. vesicatoria in host-parasite interactions

Tn5 insertion mutants of Xanthomonas campestris pv. vesicatoria were inoculated into tomato and screened for reduced virulence. One mutant exhibited reduced aggressiveness and attenuated growth in planta. Southern blot analyses indicated that the mutant carried a single Tn5 insertion not associated with previously cloned pathogenicity-related genes of X. campestris pv. vesicatoria. The wild-type phenotype of this mutant was restored by one recombinant plasmid (pOPG361) selected from a genomic library of X. campestris pv. vesicatoria 91-118. Tn3-gus insertion mutagenesis and sequence analyses of a subclone of pOPG361 identified a 1,929-bp open reading frame (ORF) essential for complementation of the mutants. The predicted protein encoded by this ORF was highly homologous to the previously reported pathogenicity-related HrpM protein of Pseudomonas syringae pv. syringae and OpgH of Erwinia chrysanthemi. Based on homology, the new locus was designated opgHXcv. Manipulation of the osmotic potential in the intercellular spaces of tomato leaves by addition of mannitol at low concentrations (25 to 50 mM) compensates for the opgHXcv mutation.     

Characterization of insertion sequence IS892 and related elements from the cyanobacterium Anabaena sp. strain PCC 7120.

IS892, one of the several insertion sequence (IS) elements discovered in Anabaena sp. strain PCC 7120 (Y. Cai and C. P. Wolk, J. Bacteriol. 172:3138-3145, 1990), is 1,675 bp with 24-bp near-perfect inverted terminal repeats and has two open reading frames (ORFs) that could code for proteins of 233 and 137 amino acids. Upon insertion into target sites, this IS generates an 8-bp directly repeated target duplication. A 32-bp sequence in the region between ORF1 and ORF2 is similar to the sequence of the inverted termini. Similar inverted repeats are found within each of those three segments, and the sequences of these repeats bear some similarity to the 11-bp direct repeats flanking the 11-kb insertion interrupting the nifD gene of this strain (J. W. Golden, S. J. Robinson, and R. Haselkorn, Nature [London] 314:419-423, 1985). A sequence similar to that of a binding site for the Escherichia coli integration host factor is found about 120 bp from the left end of IS892. Partial nucleotide sequences of active IS elements IS892N and IS892T, members of the IS892 family from the same Anabaena strain, were shown to be very similar to the sequence of IS892.     

Identification of a nucleotide sequence conserved in Lactococcus lactis bacteriophages

A genetic element which is conserved in the genomes of numerous Lactococcus lactis bacteriophage isolates has been identified and its nucleotide sequence determined. Approximately 95-99% of all L. lactis bacteriophages collected over a period of six years from two geographically distinct sources carry this conserved DNA fragment. Genetic variation in other regions of the genomes of these bacteriophages is exhibited by changes in the overall restriction patterns. The complete nt sequence for a 1.6-kb region from nine independent L. lactis bacteriophage isolates was determined and only five changes in the nt sequence were observed within a span of 1536 bp. This region has a single large 1356-bp open reading frame (ORF) coding for a 51-kDa protein. Three out of the five changes occur in a 187-bp region, 5' to this large ORF. The two additional changes are found within the 1356-bp ORF, which results in two amino acid substitutions that do not, however, change the net charge of the protein. The encoded protein is extremely charged and shares some homology with yeast translation initiation factor. In addition, there is a potential zinc-binding domain within this protein, similar to those observed in genes from bacteriophages T4 and T7.     

ISZm1068: an IS5-like insertion element from Zymomonas mobilis

A new insertion sequence, designated ISZm1068, was isolated from Zymomonas mobilis strain CP4. This element consists of 1,068 bp and contains one major ORF which shows similarities both at the nucleotide and at the amino acid sequence level with the corresponding ORFs encoding the transposases of many IS5 family elements, in particular the IS1031 group. Moreover, the Z. mobilis ORF shares the conserved N2, N3 and C1 signature motifs of the IS4 and IS5 families. Six out of seven Z. mobilis wild-type strains were shown by hybridisation to contain a single copy of the ISZm1068 element. Nucleotide sequences of the insertion elements from these strains exhibited extremely high levels of identity, varying from 94.25 to 99.25%. ISZm1068 was shown to be active in Escherichia coli cells and led to plasmid replicon fusions within the host cell. Sequence analysis of rare cointegration and resolution derivatives suggests that ISZm1068 has putative imperfect inverted repeats at its extremities of 18 bp (IR-right) and 14 bp (IR-left), and that a 3-bp (5'-TCA-3') target sequence is duplicated upon insertion.     

Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli.

From genomic libraries of Alcaligenes eutrophus H16 in lambda L47 and in pVK100, we cloned DNA fragments which restored the wild-type phenotype to poly(beta-hydroxybutyric acid) (PHB)-leaky mutants derived from strains H16 and JMP222. The nucleotide sequence analysis of a 4.5-kb region of one of these fragments revealed two adjacent open reading frames (ORF) which are relevant for the expression of the PHB-leaky phenotype. The 1,799-bp ORF1 represented a gene which was referred to as phbI. The amino acid sequence of the putative protein I (Mr, 65,167), which was deduced from phbI, exhibited 38.9% identity with the primary structure of enzyme I of the Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system (PEP-PTS). The upstream 579-bp ORF2 was separated by 50 bp from ORF1. It included the 270-bp phbH gene which encoded protein H (Mr, 9,469). This protein exhibited 34.9% identity to the HPr protein of the E. coli PEP-PTS. Insertions of Tn5 in different PHB-leaky mutants were mapped at eight different positions in phbI and at one position in phbH. Mutants defective in phbH or phbI exhibited no pleiotropic effects and were not altered with respect to the utilization of fructose. However, PHB was degraded at a higher rate in the stationary growth phase. The functions of these HPr- and enzyme I-like proteins in the metabolism of PHB are still unknown. Evidence for the involvement of these proteins in regulation of the metabolism of intracellular PHB was obtained, and a hypothetical model is proposed.     

The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper-inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild-type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild-type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild-type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.