- Volume 144, Issue 7, 1998

Shigellosis is characterized by a strong inflammatory response which is induced by bacteria invading the colonic mucosa. Characterization of a sepA mutant indicated that SepA, the major protein secreted by Shigella flexneri growing in laboratory media, might be involved in invasion and destruction of the host intestinal epithelium. The sequence of the first 500 residues of mature SepA (110 kDa) is homologous to that of the N-terminal region of lgA1 proteases. To investigate the potential proteolytic activity of SepA, the activity of the purified protein on a wide range of synthetic peptides was tested. SepA hydrolysed several of these substrates and the activity was inhibited by PMSF. Several peptides which were hydrolysed by SepA have been described as specific substrates for cathepsin G, a serine protease produced by polymorphonuclear leukocytes that was proposed to play a role in inflammation. However, unlike cathepsin G, SepA degraded neither fibronectin nor angiotensin I and had no effect on aggregation of human platelets. In addition, analysis of SepA hydrolysis by proteinase K suggested that the protein is composed of two domains of about 450 residues separated by a hinge region of 100 residues. The 47 kDa N-terminal domain was stable and endowed with proteolytic activity.

An excentric position of the nuclei, random orientation of mitoses, and multinuclear budding cells were identified in part of a population of temperature-sensitive (ts) Saccharomyces cerevisiae actin mutants at the permissive temperature of 23 ° by fluorescence and electron microscopy. The phenotype resembled that of mutants in -tubulin, dynein, JNM1, NUM1, ACT3, ACT5, myosins, profilin, tropomyosin 1, SLA2 and other genes. The question was addressed whether the cause was (i) defects in cell polarity in some ts actin mutants, manifested by lack of asymmetry of actin cortical patches, or (ii) lack of cytoplasmic or astral microtubules. The results indicated that in the cells with the nuclear defects, actin cortical patches showed the normal asymmetric distribution typical of undisturbed polarity. Cytoplasmic, astral and spindle microtubules were also preserved. The principal difference found between the wild-type and actin mutant cells was in actin cables, which in the actin mutants were developed insufficiently. It is suggested that actin cables serve as a ‘suspensory apparatus’ and/or ‘intracellular corridor’, predetermining: the location of the nucleus in the central position in interphase; the axis of nuclear movement to the bud neck before mitosis; the direction of the elongating nucleus during mitosis; and the motion of each nucleus from an excentric to a central position during cytokinesis, in cooperation with the above-mentioned and other gene products.

In strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis. The argC reported from Anabaena sp. strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1. The newly identified gene from N. ellipsosporum was denoted argL. The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine. Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid. However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.

To investigate the metabolism of (p)ppGpp in amino-acid-producing coryneform bacteria, a PCR-based strategy using degenerate consensus oligonucleotides was applied to isolate the rel gene of Corynebacterium glutamicum ATCC 13032. The gene consists of 2283 nucleotides and encodes a protein of 760 amino acids with a molecular mass of 84.4 kDa. The amino acid sequence revealed extensive similarities to the related proteins RelA and SpoT of Escherichia coli, which are known to be involved in (p)ppGpp biosynthesis and degradation. The C. glutamicum rel gene is located downstream of the apt gene encoding an adenine phosphoribosyltransferase, and an ORF with similarities to dciAE, which represents part of a dipeptide transport system in E. coli. A C. glutamicum mutant strain carrying a defined deletion in the rel gene was constructed. This mutant failed to accumulate (p)ppGpp in response to amino acid starvation. When overexpressed in E. coli, the C. glutamicum rel gene was able to reverse growth defects caused by an overexpressed relA gene. It is proposed that the C. glutamicum rel gene encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp-degrading activities.

A simple system has been developed for generating Corynebacterium glutamicum strains containing stable replicative plasmids integrated into the chromosome via homologous recombination. The system is based upon extremely strong incompatibility between two plasmids, which cannot be comaintained even under antibiotic selective pressure. Integration of the resident plasmid that contained the trpD gene of C. glutamicum was achieved by introduction of a second plasmid and subsequent selection for the maintenance of both plasmids. Plasmid integrates positive for both plasmid markers were obtained at a frequency about 10-3 of the normal transformation frequency with selection for the maintenance of only the second plasmid. Southern analysis revealed that the integration had occurred through a single-crossover homologous recombination between the trpD regions of the host genome and the plasmid. On the basis of the Campbell-type integration, chromosome walking was attempted by using Escherichia coli replication origins that were also present in the integrated plasmid. The chromosomal DNA was digested, ligated, and used to transform E. coli, which enabled recovery of the expected adjacent genomic DNA regions. The plasmid integrate was stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated sequences carrying a replicon active in the host were maintained as a stable chromosomal insert in C. glutamicum.

A single Borrelia burgdorferi bacterium may contain six or more different 32 kb circular plasmids (cp32s). Although these plasmids are homologous throughout much of their sequences, two loci have been identified at which they can vary significantly. The cp32 plasmids and their relatives each contain two adjacent genes, orfC and orf3, that vary in sequence between plasmids found within clones of individual bacteria. The orfC gene product is homologous to proteins involved in partitioning of bacterial plasmids, and the differences at this locus between plasmids may account for their compatibility. The orfC-orf3 loci are located approximately 5 kb from another variable locus called erp. The orfC-orf3 loci were used as physically linked markers to assess genetic rearrangements in the erp loci; this revealed examples of recombination involving both individual genes and entire erp loci. Recombination of the genes encoding the Erp antigens might contribute to the evasion of the mammalian immune response and could play roles in the establishment and persistence of B. burgdorferi infections in mammalian hosts.

The sequence of the dsr gene region of the phototrophic sulfur bacterium Chromatium vinosum D (DSMZ 180T) was determined to clarify the in vivo role of ‘reverse’ sirohaem sulfite reductase. The dsrAB genes encoding dissimilatory sulfite reductase are part of a gene cluster, dsrABEFHCMK, that encodes four small, soluble proteins (DsrE, DsrF, DsrH and DsrC), a transmembrane protein (DsrM) with similarity to haem-b-binding polypeptides and a soluble protein (DsrK) resembling [4Fe---4S]-cluster-containing heterodisulfide reductase from methanogenic archaea. Northern hybridizations showed that expression of the dsr genes is increased by the presence of reduced sulfur compounds. The dsr genes are not only transcribed from a putative promoter upstream of dsrA but primary transcripts originating from (a) transcription start site(s) downstream of dsrB are also formed. Polar insertion mutations immediately upstream of dsrA, and in dsrB, dsrH and dsrM, led to an inability of the cells to oxidize intracellularly stored sulfur. The capability of the mutants to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic conditions was unaltered. Photoorganoheterotrophic growth was also unaffected. ‘Reverse’ sulfite reductase and DsrEFHCMK are, therefore, not essential for oxidation of sulfide or thiosulfate, but are obligatory for sulfur oxidation. These results, together with the finding that the sulfur globules of C. vinosum are located in the extracytoplasmic space whilst the dsr gene products appear to be either cytoplasmic or membrane-bound led to the proposal of new models for the pathway of sulfur oxidation in this phototrophic sulfur bacterium.

Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

A novel filamentous bacteriophage, fs-2, was isolated from Vibrio cholerae O139 strain MDO14. The fs-2 phage was a long filamentous particle 1200 nm long and 7 nm wide. The purified phage formed a turbid plaque when spotted on a lawn of the host organisms. The plaque-formation activity was stable following heating to 70 � but was inhibited by treatment with chloroform. fs-2 had a single-stranded DNA genome and was converted to a double-stranded replicative form in the host cell. Almost all V. cholerae O139 and O1 El Tor biotype strains tested were sensitive to the phage, but most O1 classical strains and non-O1 non-O139 strains were resistant. The fs-2 genome comprised 8651 nucleotides containing nine open reading frames, five of which had predicted protein products partially homologous to the reported protein products of other filamentous phages. Although the extent of the homology was not particularly high, the genetic organization of other filamentous phages appears to be preserved in fs-2. The phage was not integrated into the chromosome of its host, but a 715 nucleotide fragment located in the large intergenic region of fs-2 was highly homologous to a part of region RS2 (repetitive sequence 2) of the V. cholerae CTX sequence which is speculated to be required for integration of the phage into the V. cholerae chromosome at a specific site.

RNA decay in bacteria is carried out by a number of enzymes that participate in the coordinated degradation of their substrates. Endo- and exonucleolytic cleavages as well as polyadenylation are generally involved in determining the half-life of RNAs. Small, untranslated antisense RNAs are suitable model systems to study decay. A study of the pathway of degradation of CopA, the copy number regulator RNA of plasmid R1, is reported here. Strains carrying mutations in the genes encoding RNase E, polynucleotide phosphorylase (PNPase), RNase II and poly(A) polymerase I (PcnB/PAP I) -- alone or in combination -- were used to investigate degradation patterns and relative half-lives of CopA. The results obtained suggest that RNase E initiates CopA decay. Both PNPase and RNase II can degrade the major 3-cleavage product generated by RNase E. This exonucleolytic degradation is aided by PcnB, which may imply a requirement for A-tailing. RNase II can partially protect CopA's 3′-end from PNPase-dependent degradation. Other RNases are probably involved in decay, since in rnblpnp double mutants, decay still occurs, albeit at a reduced rate. Experiments using purified RNase E identified cleavage sites in CopA in the vicinity of, but not identical to, those mapped in vivo, suggesting that the cleavage site specificity of this RNase is modulated by additional proteins in the cell. A model of CopA decay is presented and discussed.

Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. The binding domain of the FimH adhesin has been studied by constructing hybrids of FimH and a homologous protein, FocH, originating from F1C fimbriae. F1C fimbriae do not bind to D-mannosides or confer agglutination of any known types of erythrocytes or yeast. It was previously shown that the FocH protein can be readily substituted by the FimH adhesin, resulting in hybrid fimbriae with the same binding characteristics as type 1 fimbriae. The receptor binding of fimbriae-presented chimeric FimH--FocH hybrids was studied. FimH--FocH fusions encompassing 72% of the N-terminus of FimH fused to the complementary sector of FocH conferred agglutination of erythrocytes and yeast cells at a comparable level to FimH. Surprisingly, it was also found that similar fusions containing between 56 and 66% FimH still conferred binding to yeast cells, D-mannose--BSA and D-mannose--beads but did not give rise to agglutination. The receptor binding capacity of fusions containing 50% or less of the FimH N-terminal region was virtually abolished. The results point to the presence of a D-mannose-receptor-binding core domain in FimH, the affinity of which is modulated by other sectors of the protein to enable binding to extended mannose-containing targets.

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.

Methyl-accepting chemotaxis proteins (MCPs) play important roles in the chemotactic response of many bacteria. Oligonucleotide primers designed to amplify the conserved signalling domain of MCPs by PCR were used to identify potential MCP-encoding genes in Rhizobium leguminosarum. Using a PCR-derived probe created from these primers a genomic library of R. leguminosarum VF39SM was screened; at least five putative MCP-encoding genes (termed mcpB to mcpF) were identified and isolated from the library. One of these putative genes (mcpC) is located on one of the indigenous plasmids of VF39SM. Fifteen different cosmids showing homology to an mcpD probe were also isolated from a genomic library. The complete DNA sequences of mcpB, mcpC and mcpD were obtained. All three genes code for proteins with characteristics typical of MCPs. However, the protein encoded by mcpB has a relatively large periplasmic domain compared to that in other MCPs. Partial DNA sequences of mcpE and mcpF had strong similarity to sequences from the methylation domains of known MCPs. Mutants defective in mcpB, mcpC, mcpD or mcpE were created using insertional mutagenesis strategies. Mutation of mcpB resulted in impairment of chemotaxis to a wide range of carbon sources on swarm plates; phenotypes for the other three mutants have yet to be elucidated. The mcpB, mcpC and mcpD mutants were tested for loss of nodulation competitiveness. When co-inoculated with the wild-type, the mcpB and mcpC mutants formed fewer nodules than the wild-type, whereas the mcpD mutant was just as competitive as the wild-type. The results overall suggest that R. leguminosarum possesses mcp-like genes, and that at least some of these play a role in early steps in the plant-microbe interaction.

The motile TnphoA mutant IC24 of Proteus mirabilis U6450 generates an aberrant swarming colony, and was shown to be impaired in swarm cell differentiation, i.e. cell elongation and hyperflagellation, causing delayed and slower population migration across a solid growth medium. Levels of transcript from the flagellin filament gene fliC, the flagellar master operon flhDC, and the leucine-responsive regulatory protein gene Irp, a regulator of swarming differentiation, were reduced in IC24 mutant swarm cells. The transposon had inserted into a gene encoding a putative P-type ATPase closely related to those transporting cations across bacterial membranes. This ppa gene (Proteus P-type ATPase) was maximally expressed in differentiated swarm cells. The data suggest an effect of ion homeostasis on swarm cell differentiation, possibly mediated via the Irp--flhDC pathway.