The genetics and biochemistry of urease in Ustilago violacea

Two complementing loci in different linkage groups of the basidiomycete Ustilago violacea are involved in urease activity: a structural one (ure-1) and a second inferred to involve a permease (ure-2) locus. Two types of complementing mutations occur in the structural locus: null activity (ure-1a) and obviously reduced activity (ure-1b). The ure-2 mutants lacked urease activity in vivo on the phenol red-urea test medium, but gave extracts with wild-type activity. Extracts from wild-type strains gave one site of urease activity after polyacrylamide gel electrophoresis. A number of ure-1b mutants and active revertants from ure-1a mutants yielded electrophoretically variant urease sites. The results are discussed in terms of enzyme polymorphism in haploid eukaryotes by one (missense) or two (null, then missense) mutations.     

Urease-Encoding Genes in Ammonia-Oxidizing Bacteria

Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the β-proteobacterium Nitrosospira sp. strain NpAV and the γ-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several β-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from β-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other β-proteobacteria. Instead, it appears that urease in β-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of γ- and β-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from β-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB.     

New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP β-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P_lysA promoter with that of the P_blaP β-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.     

Biotyping of Enterotoxigenic Staphylococcus aureus by Enterotoxin Gene Cluster (egc) Polymorphism and spa Typing Analyses

Thirty-five Staphylococcus aureus strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the egc operon and spa typing. Nineteen spa types and seven different egc operons, including four putative new egc variants, were revealed. In 13 strains, allelic variants of sei and/or seg were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a sei variant, called the sei variant, was detected in six strains. In addition, the prototype sei was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described seg variant, also exhibiting several nucleotide exchanges. spa typing was more effective than REA egc grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same spa type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing egc-positive S. aureus strains.     

Multi-Virulence-Locus Sequence Typing of Listeria monocytogenes

A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulence-associated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes.     

Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA.     

Transport activity of FhuA, FhuC, FhuD, and FhuB derivatives in a system free of polar effects, and stoichiometry of components involved in ferrichrome uptake

The Escherichia colifhu operon, composed of the fhuA, C, D, and B genes, is essential for the utilization of ferric siderophores of the hydroxamate type and for the uptake of the antibiotic albomycin. We have had difficulty studying the effects of missense mutations in individual plasmid-encoded transport genes because appropriate test strains were not found: all isolated chromosomal mutations in either one of the fhu genes (with a complete loss of function) negatively influenced the expression of other fhu genes in the operon. In order to analyze Fhu mutant proteins in a system free of polar effects, we constructed a plasmid-encoded gene cassette system by introducing unique restriction sites that allowed precise cloning of individual fhu genes. The fhu cassette operon expressed in a chromosomal fhu deletion mutant enabled us to evaluate the transport activity of mutated FhuA, FhuC, FhuD or FhuB derivatives. In addition, we found that transport across the outer membrane (via FhuA, TonB, ExbB, D) rather than transport across the cytoplasmic membrane (via FhuC, D, B) was rate limiting. The stoichiometry of the components involved in the uptake of iron(III) hydroxamates seems to be important for proper functioning. Received: 20 October 1997 / Accepted: 22 December 1997     

The narA Locus of Synechococcus sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes

The narA locus required for nitrate reduction in Synechococcus sp. strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis. The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog. Overexpression of the Synechococcus moaC gene in E. coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E. coli. Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene. Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium.     

Isolation and characterization of regulatory mutations affecting the expression of the guaBA operon of Escherichia coli K-12

Summary We isolated strains of Escherichia coli K 12 in which the lac structural genes were fused to the structural genes of the guaBA operon. These strains were used to isolate regulatory mutations that increased the expression of the guaBA operon under normal repressing conditions as compared to the wild type parental fusion strain. Three classes of guaBA specific regulatory mutations were identified. Class I regulatory mutations were trans-acting and unlinked to the guaBA operon as shown by bacteriophage P1 transduction. Class II regulatory mutations were tightly linked to the guaBA operon, cis-dominant to the wild type allele in a cis-trans analysis and were regarded as control region mutations. Class III regulatory mutations were tightly linked to the guaBA operon and trans-recessive to the wild type allele in a cis-trans analysis. We have designated the locus responsible for the class III regulatory mutations as guaR. The guaR locus is tightly linked and was mapped to the counterclockwise side of the guaBA operon. The guaR locus is proposed to specify a trans acting regulatory element involved in the regulation of the guaBA operon.     

Dissolution of Xylose Metabolism in Lactococcus lactis

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl⁻. Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl⁺ strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.     

Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer.     

Evolution of a New Gene Substituting for the leuD Gene of Salmonella typhimurium: Characterization of supQ Mutations

Alpha-isopropylmalate isomerase, the second specific enzyme in the biosynthesis of leucine, is coded for by two genes, leuC and leuD. Leucine auxotrophs, harboring leuD mutations including a deletion of the entire leuD gene, revert to leucine prototrophy owing to mutations at a locus, supQ, substantially distant to the leucine operon. A large number of independently isolated supQ mutations were characterized. A significant increase in the spontaneous frequency of supQ mutations was found after mutagenesis with 2-aminopurine, N-methyl-N′-nitro-N-nitrosoguanidine, diethyl sulfate, and nitrous acid. The supQ function in most of these strains is temperature sensitive, resulting in more efficient suppression with decreasing temperature. At higher temperatures, the supQ limits the growth rate of leuD supQ mutant strains. All supQ mutations are co-transducible with proA and proB, with co-transduction frequencies ranging from 5.4 to 99.9% for different supQ mutations. Many supQ mutations were isolated, especially after nitrous acid mutagenesis, that had acquired a simultaneous proline requirement. The data support the idea of two genes, supQ and newD, whose protein products form a complex. The newD gene product, without any genetic alteration, is capable of substituting for the missing leuD protein. However, mutations in the supQ gene (point mutations or deletions) are necessary to make the newD protein available, which is normally tied up in a complex with the supQ protein.     

Salt Tolerance in Astragalus cicer Microsymbionts: The Role of Glycine Betaine in Osmoprotection

In this study, we have investigated intrinsic salt tolerance of Astragalus cicer microsymbionts (USDA3350, ACMP18) and the role of exogenous glycine betaine in osmoprotection in these bacteria. Salt stress was imposed by NaCl concentrations ranging from 0.5 to 2 %. A. cicer mesorhizobia were capable of tolerating up to 2 % sodium chloride with a population count that was inversely proportional to the salt content. When the extracellular concentration of NaCl was raised to 2 %, the generation time of the UDSA3350 strain in the mid-exponential phase of growth was 3.9-times greater than that in the no-salt control medium, whereas the ACMP18 strain survived under the same conditions but did not multiply. Application of 1 mM glycine betaine into the salt-stressed rhizobium cultures increased the number of culturable bacteria, pointing out that this molecule was involved in restoration of osmotic balance. The decline in A. cicer symbiont viability in the medium with sodium chloride and the osmoprotective role of glycine betaine for these bacteria was confirmed in the experiment using the live/dead Bac Light Bacterial Vibility Kit. Data presented in this study showed the presence of proU-like genes in the genomes of A. cicer rhizobia with high sequence similarity to the genes of the ProU-like system in Sinorhizobium meliloti and the proU operon of Escherichia coli.