A TnphoA insertion within the Bradyrhizobium japonicum sipS gene, homologous to prokaryotic signal peptidases, results in extensive changes in the expression of PBM-specific nodulins of infected soybean (Glycine max) cells

Bradyrhizobium japonicum mutant 132 was obtained by random TnphoA mutagenesis of strain 110spc4. A 6.5 kb BamHI kanamycin-resistance-coding DNA fragment of mutant 132 was used as a hybridization probe to clone the corresponding wild-type fragment. DNA sequence analysis of a 3213 bp BamHI—ClaI fragment revealed that three open reading frames (ORFs) were encoded in the same orientation. Based on sequence similarities to other proteins in the database, the second ORF was called sipS (signal peptidase). The TnphoA insertion in mutant 132 was found to be in frame near the 3′ end of sipS. The resulting SipS—PhoA hybrid polypeptide was shown to be expressed in free-living B. japonicum and in Escherichia coli cultures. An immunoblot analysis with a polyclonal antibody directed against the alkaline phosphatase revealed the appearance of a weak signal of a 70 kDa polypeptide both in B. Japonicum and in E. coli, in agreement with the calculated size of the hybrid polypeptide. A much stronger 52 kDa band was also detected. Mutant 132 was specifically disturbed in the interaction with soybean (Glycine max) when the bacteria were released from the infection threads. The bacteroids were not stably maintained within the symbiosome. Numerous vesicles were found in the plant cytosol, which finally resulted in the formation of large vacuoles within the infected nodule cells. Immunohistochemical analyses with antibodies directed against nodulins of the peribacteroid membrane indicated a lower expression of these proteins. The mutant phenotype was genetically complemented by 4.4 kb BamHI fragment including sipS. Subfragments thereof also complemented a temperature-sensitive E. coli lepB mutant, demonstrating that the B. japonicum fragment was functionally replacing Lep^ts in E. coli. Genetic studies suggested that the three genes are organized in one common operon which is expressed from a promoter upstream of the sequenced region. Inactivation of the gene downstream of sipS did not result in a detectable phenotype.     

Use of Tn KPK2 for sequencing a 10.6-kb PstI DNA fragment of Bradyrhizobium japonicum and for the construction of aspA and ndvA mutants

Transposon TnKPK2 was used to saturate a randomly cloned Bradyrhizobium japonicum PstI fragment and the insertions were used as starting points for the sequence determination. The first gene of the 10.6-kb DNA insert encodes a homologue to ndvA, the product of which is known to be involved in the formation of periplasmic cyclic glucans. Selected TnKPK2 insertions were introduced into the B. japonicum wild-type strain. The resulting mutants were subsequently tested for their symbiotic interactions with soybeans. As in Sinorhizobium meliloti, a B. japonicum ndvA mutant was affected in salt-stress tolerance and exhibited symbiotic defects in that it induced the formation of ineffective soybean nodules. The central nodule tissue was infected by bacteroids, but within the infected cells the mutant was not properly maintained. Another gene was found to be highly similar to bacterial aspartases and thus was named aspA. The putative function of the product of this gene was confirmed by genetic complementation of aspartase-less Escherichia coli strain TK237. The symbiotic phenotype of a B. japonicum aspATnKPK2 mutant consisted of enlarged symbiosomes that made the system ineffective. In general, TnKPK2 is a suitable means for fast sequencing. In combination with pJQ200SK, the resulting recombinant plasmids can be directly used to create genetically defined mutants.     

The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase.

Interspecific complementation of a Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.     

Cloning of the gene encoding avermectin B 5-O-methyltransferase in avermectin-producing Streptomyces avermitilis

Complementation of a mutant lacking avermectin B 5-O-methyltransferase (AveD) of Streptomyces avermitilis, which catalyses the methylation of the hydroxyl group at the C5 position of avermectin B compounds, revealed that the gene encoding AveD is in a 1.25-kb SalI–EcoNI fragment in the left region of the gene cluster for avermectin biosynthesis. The nucleotide sequence of this fragment predicted a 283-aa gene product homologous to several methyltransferases requiring S-adenosyl-l-methionine as a cofactor. After cloning of the aveD region from mutant not producing AveD, the complementation experiments were performed using a pair of hybrid fragments (AveD⁺/AveD⁻ and AveD⁻/AveD⁺). They suggest that the mutation(s) is in the N-terminus of AveD. SSCP analysis of amplified DNA of the aveD region derived from both wild type and mutant strains supports the results of the complementation experiments. Sequence analysis of the aveD region of the mutant strain revealed that a point mutation is within ORF, being Thr23→Ile substitution. This mutation causes the inactivation of O-methyltransferase activity of AveD.     

Mutation in the lysA gene impairs the symbiotic properties of Mesorhizobium ciceri

A Tn5-induced mutant of Mesorhizobium ciceri, TL28, requiring the amino acid lysine for growth on minimal medium was isolated and characterized. The Tn5 insertion in the mutant strain TL28 was located on a 6.8-kb EcoRI fragment of the chromosomal DNA. Complementation analysis with cloned DNA indicated that 1.269 kb of DNA of the 6.8-kb EcoRI fragment restored the wild-type phenotype of the lysine-requiring mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to lysA gene of different bacteria. The lys ^? mutant TL28 was unable to elicit development of effective nodules on the roots of Cicer arietinum L. There was no detectable level of lysine in the root exudates of chickpea. However, addition of lysine to the plant growth medium restored the ability of the mutant to produce effective nodules with nitrogen fixation ability on the roots of C. arietinum.     

DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δrtx::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.     

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli

Summary A system is described which enabled the selection of a heterologous ep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36000 daltons, which is in agreement with the calculated molecular weight of 35782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I gen/es.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.     

A Bacillus subtilis locus encoding several gene products affecting transport of cations

A 3.6-kb DNA fragment from Bacillus subtilis was found to complement the K⁺ uptake-deficient Escherichia coli strain TK2420. Transformation with a pKLO61 plasmid harboring this fragment conferred the capacity to grow on a minimal medium containing only 10 mM K⁺. Insertional mutagenesis and subcloning identified a single gene responsible for the complementation. This gene coded for an apparent homolog of E. coli TrkA. Sequence analysis of the cloned region also revealed three additional open reading frames. These included: a gene encoding a homolog to the czcD gene product of Alcaligenes eutrophus, a lysR-type regulatory gene which was found to enhance Na⁺ resistance in E. coli NM81 (ΔnhaA) in a separate complementation test, and an orfD with no significant similarity to sequences deposited in Genbank.     

Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane α-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min^–1 mg (dry mass)^–1 as compared to the wild-type strain rate of 1.2 nmol min^–1 mg (dry mass)^–1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min^–1 mg (dry mass)^–1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake. Received: 16 October 1997 / Accepted: 27 November 1997     

Cloning,sequencing, and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene

Bacillus sp. B-0618 produces both creatinase (Cre; creatine amidinohydrolase; EC 3.5.3.3) and sarcosine oxidase (Sox; EC 1.5.3.1) enzymes when grown in the presence of an inducer, choline chloride. A genomic library of Bacillus sp. B-0618, prepared in the plasmid vector pACYC184, was screened to obtain a gene (sox) encoding Sox by a convenient colorimetric assay. A plasmid, pOXI101, isolated from a sox-positive clone, contained a 14.2-kb insert of Bacillus DNA. The nucleotide sequence of a 1.7-kb segment containing the sox gene was determined, and it was found that an open reading frame encoding a protein consisting of 390 amino acids was located upstream from the cre structural gene cloned previously. When a 1.6-kb XhoI-BglII fragment of pOXI101 was inserted into the pUC118 vector and introduced into Escherichia coli, transformants cultured in the absence of the inducer produced Sox about 50-fold more than Bacillus sp. B-0618 cultured in the presence of the inducer. The Bacillus Sox had the -¹¹Gly-X-¹³Gly-X-X-¹⁶Gly- sequence motif that is highly conserved in flavoproteins. We created an FAD-free Sox by changing ¹³Gly to Asp in the motif of the parental Sox by oligodeoxynucleotide-directed mutagenesis. The mutant protein no longer expressed the Sox activity, even on the addition of FAD.     

An unusual suicidal interaction inEscherichia coli involving nucleoid protein H-NS

A conditional-lethal mutation (rpoB364) mapping to the gene that encodes the β-subunit of RNA polymerase was obtained inEscherichia coli. This mutation caused cell filamentation at the restrictive growth temperature and partial derepression of the osmotically regulatedproU operon at the permissive growth temperature. Even under the latter condition, transformants of therpoB364 mutant strain carrying the plasmid vector pACYC184, but not those carrying otherpolA-dependent multicopy plasmids such as pACYC177 or pBR322, were killed in early stationary phase; one class of suppressor mutants isolated as survivors within these transformant colonies were further derepressed forproU-lac expression, and the mutation in each of several independent clones of this class was mapped tohns, the gene that encodes the protein H-NS of theE. coli nucleoid. Thehns mutations did not suppress the conditional-lethal growth phenotype of therpoB364 mutant itself. On the other hand, intracellular overproduction of guanosine 3’, 5’-bispyrophosphate (ppGpp) in therpoB364 strain alleviated both the growth inhibition at the restrictive temperature and the pACYC184-mediated stationary-phase lethality. Upon subcloning into pUC19 or into pACYC177, a 105-bpXbal-HindIII fragment from pACYC184 was shown to be sufficient to confer therpoB364 hns ⁺-dependent lethal phenotype. We suggest that the level in stationary-phase cultures of a gene product(s) that interacts with the pACYC184 DNA fragment is altered in therpoB364 hns+derivative (compared to that inrpoB+ orrpoB364 hns strains) and that this results in cell suicide.     

Primary structure of the hip gene of Escherichia coli and of its product,the β subunit of integration host factor

We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the β subunit of integration host factor (IHFβ). In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip⁺ and which hip^? by complementation, and then sequenced the ends of the critical deletions. The 5′ end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF. Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations. We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3′ end of the neighboring gene. rpsA, and we constructed an IHFβ overproducer by fusing the coding sequences to the λp_L promoter. A survey of known protein sequences revealed a close relationship between IHFβ and the type II prokaryotic DNA binding proteins (the “histone-like” proteins). This relationship is shared to a considerable extent by the other subunit of IHF, IHFα. A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.     

Cloning and characterisation of the Neisseria gonorrhoeaearoB gene

The gene coding for the 3-dehydroquinate synthetase (aroB) of Neisseria gonorrhoeae has been cloned by functional complementation of an Escherichia coli aroB mutant. The aroB gene isolated from a gonococcal plasmid library encodes a 359 amino acid protein with a molecular mass of 38.6?kDa. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 33 to 55%. An open reading frame coding for an aroK homologue is located immediately upstream of aroB. Downstream of aroB a region of inverted repeats and a gene showing high homology to yafJ of E. coli has been identified. Disruption of aroB generates a gonococcal mutant that is unable to grow in the absence of aromatic compounds. Complementation of the mutant with the intact aroB gene intrans indicates that the gene is responsible for the auxotrophic phenotype. In infection assays with AroB-deficient gonococcal strains, binding, entry and short-term survival in epithelial cells is not affected. The aroB gene might be useful as a selectable marker and target for attenuation of a gonococcal live vaccine strain or as a biosafe laboratory strain.     

A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

Nutritional iron acquisition by bacteria is well described, but almost nothing is known about bacterial iron export even though it is likely to be an important homeostatic mechanism. Here, we show that Bradyrhizobium japonicum MbfA (Blr7895) is an inner membrane protein expressed in cells specifically under high iron conditions. MbfA contains an N-terminal ferritin-like domain (FLD) and a C-terminal domain homologous to the eukaryotic vacuolar membrane Fe²⁺/Mn²⁺ transporter CCC1. An mbfA deletion mutant is severely defective in iron export activity, contains >2-fold more intracellular iron than the parent strain, and displays an aberrant iron-dependent gene expression phenotype. B. japonicum is highly resistant to iron and H₂O₂ stresses, and MbfA contributes substantially to this as determined by phenotypes of the mbfA mutant strain. The N-terminal FLD was localized to the cytoplasmic side of the inner membrane. Substitution mutations in the putative iron-binding amino acid residues E20A and E107A within the N-terminal FLD abrogate iron export activity and stress response function. Purified soluble FLD oxidizes ferrous iron (Fe²⁺) to incorporate ferric iron (Fe³⁺) in a 2:1 iron:protein ratio, which does not occur in the E20A/E107A mutant. The FLD fragment is a dimer in solution, implying that the MbfA exporter functions as a dimer. MbfA belongs to a protein family found in numerous prokaryotic genera. The findings strongly suggest that iron export plays an important role in bacterial iron homeostasis.     

Cloning and characterization of the groE locus from Actinobacillus pleuropneumoniae

A 4.4-kb DNA fragment was cloned from Actinobacillus pleuropneumoniae (strain 4074, serotype 1) by genetic complementation with Escherichia coli groES-groEL mutant strains. Sequence analysis of this fragment revealed a purine nucleoside phosphorylase (DeoD)-encoding gene homolog (deoD), heat-shock response-encoding genes for the small (groES) and large subunits (groEL) and a partial open reading frame encoding an alcohol dehydrogenase homolog (adhE). The predicted amino-acid sequence of groES and groEL genes showed extensive sequence identity (80–95%) with other Pasteurellaceae. The gene organization surrounding the groE locus was different from that of Haemophilus infuenzae. When expressed in E. coli, groES-groEL genes were capable of complementing the growth of a λ lytic phage, indicating a structural as well as functional conservation.