- Volume 147, Issue 11, 2001

Yersinia enterocolitica serotype O9 may cause a persistent intestinal infection with few or no symptoms in humans and in BALB/c mice. The present study demonstrated profound alterations in the immune status of BALB/c mice infected with Y. enterocolitica O9. Infected mice developed splenomegaly and phenotypic analysis of spleen cells revealed increases in CD3+ total T cells, CD4+ helper T cells, CD8+ cytotoxic T cells and CD11b+ phagocytic cells. Spleen cells from infected mice exhibited impaired responses to mitogens and suppressed the proliferation of normal splenocytes in response to mitogens. Suppression of responses to concanavalin A and heat-killed yersiniae was associated with increased production of gamma interferon and reactive nitrogen intermediates. Y. enterocolitica-infected mice resisted challenge with a lethal dose of the intracellular pathogen Listeria monocytogenes. These findings suggest that infection of mice with Y. enterocolitica O9 induces gamma-interferon-secreting cells that promote macrophage activation, mediating resistance to infection with L. monocytogenes, and macrophage production of reactive nitrogen intermediates, which results in in vitro inhibition of lymphocyte response to mitogens.

Two genes in the Escherichia coli genome, b4065 (yjcE) and b1191 (ycgO), are similar to genes encoding eukaryotic Na+/H+ exchangers. Mutants were constructed in which yjcE (GRN11), ycgO (GRF55) or both (GRD22) were inactivated. There was no change in respiration-driven Na+ efflux in any of the mutants when grown in media containing 50–500 mM Na+. The only striking finding was that growth of GRF55 was impaired at low osmolarity. In complex low-salt medium, GRF55 grew at a wild-type rate for three to four generations but then stopped; the growth was partially recovered after a pause, the length of which was dependent on salt concentration. Measurement of cytoplasmic alkali cations showed that an abrupt loss of about one-half of the intracellular K+ preceded the pause. When grown in low-salt medium with only 20 mM added Na+, GRF55 also lost the ability to maintain a sodium concentration gradient. However, this phenomenon appears to be a secondary effect of the ycgO deletion. The double mutant GRD22 has the same properties as GRF55; no additional effect was found. The data indicate that neither ycgO nor yjeE participates in respiration-driven Na+ extrusion. Instead, ycgO is required for growth at low osmolarity. Hence it is concluded that ycgO participates in cell volume regulation, and accordingly it is suggested that ycgO be renamed cvrA.

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.

Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6·7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

WecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate N-acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in Escherichia coli and other enteric bacteria. WecA functions as an UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279–282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH279–282 and the Arg265 are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the E. coli wecA mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H279) of the HIHH motif as well as the Arg265 residue. The functional importance of these residues is underscored by the high level of conservation of H279 and Arg265 among bacterial WecA homologues that utilize several different UDP-N-acetylhexosamine substrates.

Nine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745. Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively. Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745. Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA. Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities. Wild-type mating efficiency was 10−6 transconjugants per donor, while the mutant strain yielded no transconjugants. Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10−6, while VT747 was unable to mobilize this plasmid. These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport. However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747. While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both Act. actinomycetemcomitans strains VT745 and VT747.

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying β-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2·3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess ∼∼28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.

Eleven different cyanobacteria were investigated with respect to their capabilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of poly-β-hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis using a phaC-specific DNA probe, (ii) Western blot analysis using specific polyclonal anti-PhaE antibodies raised in this study against PhaE of Synechocystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR products using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provide evidence for the presence of a type-III PHA synthase in the following cyanobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis fritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains PCC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The screening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive control, which is so far the only cyanobacterium for which molecular data of the PHA synthase genes are available. No evidence for the presence of a type-III PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 8955 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural genes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 and Chlorogloeopsis fritschii PCC 6912, and in addition a central region of the phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.

The Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by the upstream determinant rgg. In the present study, two ORFs, transcribed on the opposite DNA strand, were identified immediately downstream of gtfG. The first, designated dsg, shares a convergent putative transcriptional terminator with gtfG, and encodes a predicted 46 kDa transmembrane protein similar to the Yersinia enterocolitica TrsA involved in polysaccharide biosynthesis. Insertional inactivation of dsg resulted in only ∼∼60% of the parental level of glucosyltransferase activity. The 870 bp gene 5′ to dsg is similar to the gtfG regulatory determinant. Designated rggD, this rgg-like determinant downstream of gtfG encodes a putative 33·6 kDa cytoplasmic protein. Despite their sequence similarity, the functions of rgg and rggD appear specific. Strains in which rggD was insertionally inactivated and strains containing plasmid-borne rggD had parental levels of glucosyltransferase activity. Northern blot hybridization analyses showed ∼1·3 kb dsg-specific and ∼1·0 kb rggD-specific mRNA transcripts associated with this region; no polycistronic transcript was observed. Although rgg-like gene products have been demonstrated to function as positive transcriptional regulators of adjacent genes in several streptococcal species, Northern blot analysis suggested that rggD did not influence the transcription of dsg or the divergent downstream ylbN-like determinant under the conditions in the present study. Comparison of this S. gordonii chromosome region to other streptococcal genomes, which do not contain the rgg/rggD-flanked region involved in glucan synthesis, raised intriguing possibilities about the origins of this chromosomal region, and also suggested that rggD might regulate a distally located gene.

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P cer , located centrally within cer, is also required for stable plasmid maintenance. P cer is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P cer in plasmid monomers. P cer is unusual in that the −35 and −10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P cer activation involves realignment of the −35 and −10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

In the search for Type II polyketide synthases (PKSs) a DNA fragment was isolated from Streptomyces antibioticus ATCC 11891 (a producer of oleandomycin). DNA sequencing of the cloned fragment revealed six complete ORFs whose deduced products showed similarities to those of other genes known to be involved in polyketide biosynthesis. Several S. coelicolor strains mutated in different steps of actinorhodin biosynthesis (actI, actIII, actV A and actVII) were complemented by the cloned genes, suggesting that the isolated genes encode an aromatic polyketide of unknown structure and function. The cluster also contains a putative LysR-type transcriptional regulator (ORF0), which controls PKS gene expression in a heterologous host. DNA binding assays and transcriptional analysis suggest that the pathway-specific regulator for actinorhodin biosynthesis (actII-ORF4) is also involved in the expression of the cloned PKS in the host strain.

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7–37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2–DNA–formate complex was at least three times higher than that of the native protein–DNA–formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA− phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc–lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Aeromonas spp., considered as emerging opportunistic pathogens, belong to the family Vibrionaceae. Among the criteria currently used for their classification is the presence of a single FeSOD (iron-containing superoxide dismutase), which distinguishes them from Enterobacteriaceae. In this paper the cloning of the sodA and sodB genes encoding two different SODs in Aeromonas hydrophila ATCC 7966 is reported. The sodB gene encoded an FeSOD (196 amino acids, 21·5 kDa), was constitutively expressed and showed 75% homology with the E. coli FeSOD. The sodA gene encoded a protein of 206 amino acids (22·5 kDa) with MnSOD (manganese-containing SOD) activity and showed 55% homology with the Escherichia coli MnSOD. The MnSOD of A. hydrophila was detected only during the stationary phase of growth under high aeration or when induced by lack of iron. Nevertheless, paraquat had no detectable effect on its production. The amino-terminal part of the Mn-containing protein contained a putative signal sequence which could permit a periplasmic localization.