Expression of Rhizobium leguminosarum CFN42 genes for lipopolysaccharide in strains derived from different R. leguminosarum soil isolates.

Two mutant derivatives of Rhizobium leguminosarum ANU843 defective in lipopolysaccharide (LPS) were isolated. The LPS of both mutants lacked O antigen and some sugar residues of the LPS core oligosaccharides. Genetic regions previously cloned from another Rhizobium leguminosarum wild-type isolate, strain CFN42, were used to complement these mutants. One mutant was complemented to give LPS that was apparently identical to the LPS of strain ANU843 in antigenicity, electrophoretic mobility, and sugar composition. The other mutant was complemented by a second CFN42 lps genetic region. In this case the resulting LPS contained O-antigen sugars characteristic of donor strain CFN42 and reacted weakly with antiserum against CFN42 cells, but did not react detectably with antiserum against ANU843 cells. Therefore, one of the CFN42 lps genetic regions specifies a function that is conserved between the two R. leguminosarum wild-type isolates, whereas the other region, at least in part, specifies a strain-specific LPS structure. Transfer of these two genetic regions into wild-type strains derived from R. leguminosarum ANU843 and 128C53 gave results consistent with this conclusion. The mutants derived from strain ANU843 elicited incompletely developed clover nodules that exhibited low bacterial populations and very low nitrogenase activity. Both mutants elicited normally developed, nitrogen-fixing clover nodules when they carried CFN42 lps DNA that permitted synthesis of O-antigen-containing LPS, regardless of whether the O antigen was the one originally made by strain ANU843.     

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.)

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan = 4200 m²) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5 kb) and whole ribosomal DNA (5 kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5 kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5 kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.     

Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. IV. Genetic transformation using amplified genes from Djungarian hamster cells highly resistant to colchicine

DNA-mediated transfer of colchicine-resistance from Djungarian hamster DM5/7 cell line, 750-fold resistant to the drug, was studied. The resistance to colchicine of DM5/7 cells is due to amplification of the genes, possibly coding for the polypeptide p22. Both high-molecular weight DNA (presumably, chromosomal DNA) and low-molecular weight DNA (presumably, extrachromosomal DNA) effectively transferred the colchicine-resistance to Djungarian hamster and mouse cells. DNA of sensitive to colchicine but resistant to ouabain mouse cells CAK-143OuaR was not capable to transfer colchicine-resistance, but effectively transferred ouabain-resistance connected with a mutation in Na+/K+-dependent ATP-ase locus. The differences in genetic transformation with amplified p22 genes and mutant Na+/K+-dependent ATP-ase genes were revealed. All cells of 3 colchicine-resistant transformants of DM-15 cells and all 10 spontaneously derived resistant clones contain the additional chromosome 4. The role of trisomy 4 in the development of colchicine-resistance in DM-15 cells is discussed.     

Conservation of plasmid-encoded traits among bean-nodulating Rhizobium species

Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organization. In contrast, strains belonging to other bean-nodulating species seem to have acquired only the pSym plasmid from R. etli.     

A 3'' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Genetic diversity of root-nodulating bacteria isolated from pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Functional analysis of the herpes simplex virus UL42 protein.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Specific binding of lactoferrin to Aeromonas hydrophila

The subunit S1 of pertussis toxin (PT) was purified as the recombinant product BacS1 from the culture supernatant of a Bacillus subtilis strain containing a secretion vector with a DNA fragment coding for the mature subunit S1 inserted downstream of the signal sequence of the alpha-amylase gene. The method of purification was successive ion exchange and adsorption chromatography. BacS1 occurred in two forms (28 and 20 kDa) of which the truncated 20-kDa peptide was the main one in the supernatant. The truncated BacS1 was purified and shown to have the same NH2-terminus as the full-size (28 kDa) BacS1. It was also enzymatically active indicating correct conformation. The truncated BacS1 was also shown to elicit neutralizing and protective antibodies when injected into mice or rabbits.     

Variable regions of antibodies to synthetic polypeptides. II. Analysis of variable region genes encoding antibodies specific for (T,G)-A--L

Monoclonal antibodies specific for the synthetic polypeptide antigen (T,G)-A--L have been produced in two strains of mice, C57BL/10 and C3H.SW. The genes encoding the variable (V) regions of these antibodies have been studied by using the DNA hybridization technique of Southern, as well as by gene cloning and sequencing. Hybridization of DNA from 14 different cell lines with a kappa-chain probe revealed that the different cell lines used one of two different gene rearrangements to encode the recombined V region gene. There was a perfect correlation between light chain rearrangement, idiotype expression, and fine specificity. Hybridization analyses of the heavy chain revealed a more complex pattern. Seven hybridomas had the rearranged heavy chain V region genes on a 4.4 kb EcoRI restriction fragment. Others were found on restriction fragments that differed in length by several hundred base pairs. The recombined heavy chain V region genes were cloned from three different hybridoma cell lines secreting anti-(T,G)-A--L antibodies, all of which express the same idiotype and fine specificity pattern. Restriction mapping and sequencing indicate that all three utilize the same V gene, identified as the 186-2 germline gene. However, different D and J genes are used to encode each of the antibodies. In contrast to the results seen in other antigen systems, heavy chain D and J genes do not have a major influence on idiotype expression and fine specificity of antibodies to the synthetic polypeptide (T,G)-A--L.     

The primary structure of a halorhodopsin from Natronobacterium pharaonis. Structural, functional and evolutionary implications for bacterial rhodopsins and halorhodopsins

We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective pressures. Conserved and conservatively replaced amino acid residues in all three proteins identify general features essential for ion-motive bacterial rhodopsins, responsible for overall structure and chromophore properties. Comparison of the bacteriorhodopsin sequence with those of the two halorhodopsins, on the other hand, identifies features involved in their specific (proton and chloride ion) transport functions.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

The bacteriophage T4 regA gene: primary sequence of a translational repressor

The regA gene product of bacteriophage T4 is an autogenously controlled translational regulatory protein that plays a role in differential inhibition (translational repression) of a subpopulation of T4-encoded "early" mRNA species. The structural gene for this polypeptide maps within a cluster of phage DNA replication genes, (genes 45-44-62-regA-43-42), all but one of which (gene 43) are under regA-mediated translational control. We have cloned the T4 regA gene, determined its nucleotide sequence, and identified the amino-terminal residues of a plasmid-encoded, hyperproduced regA protein. The results suggest that the T4 regA gene product is a 122 amino acid polypeptide that is mildly basic and hydrophilic in character; these features are consistent with known properties of regA protein derived from T4-infected cells. Computer-assisted analyses of the nucleotide sequences of the regA gene and its three upstream neighbors (genes 45, 44, and 62) suggest the existence of three translational initiation units in this four-gene cluster; one for gene 45, one for genes 44, 62 and regA, and one that serves only the regA gene. The analyses also suggest that the gene 44-62 translational unit harbors a stable RNA structure that obligates translational coupling of these two genes.     

Inactivation of essential division genes, ftsA, ftsZ, suppresses mutations at sfiB, a locus mediating division inhibition during the SOS response in E. coli.

A dominant sfiB allele has been cloned which renders partial diploids of an sfiB + Escherichia coli host resistant to division inhibition mediated by the SOS response. Transpositional mutagenesis was used to map the position of this sfiB114 allele, carried by a plasmid pLG552 , to an approximately 0.6-kb region overlapping the coding regions for ftsA and ftsZ , two genes essential for normal division. Most Tn 1000 insertions which inactivated sfiB114 also inactivated the ftsA function and caused the disappearance of both a 47-K polypeptide and reduced levels of a 42-K polypeptide in maxi-cells carrying pLG552 . An additional insertion inactivating sfiB114 was mapped to the right of ftsA and resulted in loss of the 42-K but not the 47-K polypeptide in maxi-cells. Moreover, a 2.1-kb BamHI-EcoRI DNA fragment was subcloned which carried ftsA and coded for a 47-K polypeptide but did not carry sfiB114 and did not complement ftsZ . We conclude that sfiB114 is located within ftsZ coding for a 42-K polypeptide. Nevertheless, insertions into ftsZ coding the 47-K polypeptide suppress the sfiB114 allele by substantially reducing the synthesis of the FtsZ ( SfiB114 ) polypeptide. The level of residual FtsZ synthesis was minimal when Tn 1000 was inserted closest to the distal end of ftsA , indicating the presence of a regulatory region essential for maximal expression of ftsZ .     

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.     

Isolation and characterization of an operon involved in sulfate and sulfite metabolism in Sinorhizobium fredii

A gene cluster ORFabcd from a Sinorhizobium fredii HN01 mutant strain HSMRalpha was isolated. We showed that it was an operon involved in sulfur metabolism. Functional studies revealed that, except for ORFb, the three genes ORFa, ORFc and ORFd were involved in sulfite reduction. ORFa and ORFc were similar to the cysG and cysI from Sinorhizobium meliloti 1021 and Rhizobium etli CFN 42, respectively. ORFd encodes a conserved hypothetical protein in other bacteria. We demonstrate here, for the first time, that it was a new locus involved in sulfate assimilation in S. fredii HN01 and we designated it as cysII.     

The superinfection exclusion gene (sieA) of bacteriophage P22: identification and overexpression of the gene and localization of the gene product.

Previous work has shown that the sieA gene of Salmonella bacteriophage P22 is located between the genes mnt and 16. We cloned DNA fragments of the region into multicopy vectors and tested the transformants for mediating superinfection exclusion. Subcloning, phenotypical tests, and DNA sequencing resulted in the identification of the sieA gene. There are two possible initiation codons within one open reading frame of 492 or 480 bp. The deduced amino acid sequence leads to a hypothetical polypeptide with a calculated molecular mass of 18.8 or 18.3 kDa, respectively. According to three hydrophobic regions, all of which are long enough to span the membrane, the product of sieA should be a protein of the inner membrane of a P22-lysogenic cell of Salmonella typhimurium. The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction.