Showing papers in "Journal of Bacteriology in 1996"

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

Abstract: Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

Abstract: The live attenuated bacillus Calmette-Guerin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Multidrug efflux pumps of gram-negative bacteria.

Abstract: Gram-negative bacteria tend to be more resistant to lipophilic and amphiphilic inhibitors than gram-positive bacteria. Such inhibitors include dyes, detergents, free fatty acids, antibiotics, and other chemotherapeutic agents. This property is used in the selective enrichment of gram-negative bacteria, especially of members of the family Enterobacteriaceae, for example with MacConkey agar (containing crystal violet and bile salts), EMB agar (containing dyes), and deoxycholate agar (containing sodium deoxycholate). The fact that Escherichia coli K-12 can grow in the presence of 1% sodium dodecyl sulfate has been rediscovered many times. Many lipophilic antibiotics, such as penicillin G, erythromycin, fusidic acid, and rifamycin SV, are much less active against most gram-negative bacteria. In fact, a survey of recently reported antibiotics of natural origin showed that, among those compounds that showed activity against gram-positive bacteria, more than 90% lacked activity at a useful level against E. coli (51). This intrinsic resistance of gram-negative bacteria has often been attributed entirely to the presence of the outer membrane barrier. This barrier does contribute to the resistance, as the narrow porin channels slow down the penetration of even small hydrophilic solutes, and the low fluidity of the lipopolysaccharide leaflet decreases the rate of transmembrane diffusion of lipophilic solutes (38, 40). However, the outer membrane barrier cannot be the whole explanation, even with species such as Pseudomonas aeruginosa which produces an outer membrane of exceptionally low permeability (1, 57). This is seen from the fact that equilibration across the outer membrane is achieved very rapidly, in part because the surface-to-volume ratio is very large in a small bacterial cell. Thus, the periplasmic concentrations of many antibiotics are expected to reach 50% of their external concentrations in 10 to 30 s in P. aeruginosa and in a much shorter time period in E. coli (34). Additional mechanisms are therefore needed to explain the level of intrinsic resistance. With the earlier b-lactam compounds, this second contributing factor is the hydrolysis by the periplasmic b-lactamases that are encoded by chromosomal genes in many gram-negative bacteria, and the levels of resistance can be explained quantitatively, in many cases, by the synergy between the outer membrane barrier and b-lactamase (37). However, with dyes, detergents, other classes of antibiotics, and even the b-lactams developed more recently that are not hydrolyzed easily by common b-lactamases, the second factor was completely unknown (34). Recent studies showed that multipledrug efflux pumps, many with unusually broad specificities, play a major role in the intrinsic resistance of gram-negative bacteria. Active efflux processes have been known to cause drug resistance in gram-negative bacteria, through the pioneering studies of S. Levy (19) on Tet-mediated tetracycline resistance. Each of these traditional efflux pumps excretes only one drug or one class of drugs (35). In contrast, a multidrug efflux pump can pump out a wide range of compounds, and it is often difficult to discern any common structural features among the substrates (20, 35).

The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis.

Abstract: The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to biofilm production of Staphylococcus epidermidis, which is thought to contribute to virulence in biomaterial-related infections. We purified a specific polysaccharide antigen of biofilm-producing S. epidermidis 1457 and RP62A, which was recently shown to have a function in the accumulative phase of biofilm production by mediating intercellular adhesion (D. Mack, M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs, Infect. Immun. 62:3244-3253, 1994). Following Sephadex G-200 gel filtration, this antigen was separated by Q-Sepharose chromatography into a major polysaccharide, polysaccharide I (> 80%), which did not bind to Q-Sepharose, and a minor polysaccharide, polysaccharide II (< 20%), which was moderately anionic. As shown by chemical analyses and nuclear magnetic resonance spectroscopy, polysaccharide I is a linear homoglycan of at least 130 beta-1,6-linked 2-deoxy-2-amino-D-glucopyranosyl residues. On average, 80 to 85% of them are N acetylated; the rest are non-N-acetylating and positively charged. Chain cleavage by deamination with HNO2 revealed a more or less random distribution of the non-N-acetylated glucosaminyl residues, with some prevalence of glucosaminyl-rich sequences. Cation-exchange chromatography separated molecular species whose content of non-N-acetylated glucosaminyl residues varied between 2 and 26%. Polysaccharide II is structurally related to polysaccharide I but has a lower content of non-N-acetylated D-glucosaminyl residues and contains phosphate and ester-linked succinate, rendering it anionic. Enzyme-linked immunosorbent assay inhibition with various monosaccharides revealed the beta-anomeric form and the acetylated amino group of the D-glucosaminyl residues as important for reactivity with the specific antiserum. The unbranched polysaccharide structure favors long-range contacts and interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular adhesion and biofilm accumulation. The structure of the polysaccharide is, so far, considered to be unique and, according to its function, is referred to as S. epidermidis polysaccharide intercellular adhesin (PIA).

AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants.

Abstract: Multiple-antibiotic-resistance (Mar) mutants of Escherichia coli are resistant to a wide variety of antibiotics, and increased active efflux is known to be responsible for the resistance to some drugs. The identity of the efflux system, however, has remained unknown. By constructing an isogenic set of E. coli K-12 strains, we showed that the marR1 mutation was incapable of increasing the resistance level in the absence of the AcrAB efflux system. This experiment identified the AcrAB system as the major pump responsible for making the Mar mutants resistant to many agents, including tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin.

Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling.

Abstract: Acylated homoserine lactones (AHLs) play a widespread role in intercellular communication among bacteria. The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules. We report here that these metabolites inhibited AHL-controlled processes in prokaryotes. Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems.

Flagellin A is essential for the virulence of Vibrio anguillarum.

Abstract: A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.

Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.

Abstract: Forty-one ionizing radiation-sensitive strains of Deinococcus radiodurans were evaluated for their ability to survive 6 weeks of desiccation. All exhibited a substantial loss of viability upon rehydration compared with wild-type D. radiodurans. Examination of chromosomal DNA from desiccated cultures revealed a time-dependent increase in DNA damage, as measured by an increase in DNA double-strand breaks. The evidence presented suggests that D. radiodurans9 ionizing radiation resistance is incidental, a consequence of this organism9s adaptation to a common physiological stress, dehydration.

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

Abstract: In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.

Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes.

Abstract: Mycothiol [2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl- (1-->1)-myo-inositol] (MSH) has recently been identified as a major thiol in a number of actinomycetes (S. Sakuda, Z.-Y. Zhou, and Y. Yamada, Biosci. Biotech. Biochem. 58:1347-1348, 1994; H. S. C. Spies and D. J. Steenkamp, Eur. J. Biochem. 224:203-213, 1994; and G. L. Newton, C. A. Bewley, T. J. Dwyer, R. Horn, Y. Aharonowitz, G. Cohen, J. Davies, D. J. Faulkner, and R. C. Fahey, Eur. J. Biochem. 230:821-825, 1995). Since this novel thiol is more resistant than glutathione to heavy-metal ion-catalyzed oxidation, it seems likely to be the antioxidant thiol used by aerobic gram-positive bacteria that do not produce glutathione (GSH). In the present study we sought to define the spectrum of organisms that produce MSH. GSH was absent in all actinomycetes and some of the other gram-positive bacteria studied. Surprisingly, the streptococci and enterococci contained GSH, and some strains appeared to synthesize it rather than import it from the growth medium. MSH was found at significant levels in most actinomycetes examined. Among the actinobacteria four Micrococcus species produced MSH, but MSH was not found in representatives of the Arthrobacter, Agromyces, or Actinomyces genera. Of the nocardioforms examined, Nocardia, Rhodococcus, and Mycobacteria spp. all produced MSH. In addition to the established production of MSH by streptomycetes, we found that Micromonospora, Actinomadura, and Nocardiopsis spp. also synthesized MSH. Mycothiol production was not detected in Propionibacterium acnes or in representative species of the Listeria, Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Clostridium genera. Examination of representatives of the cyanobacteria, purple bacteria, and spirochetes also gave negative results, as did tests of rat liver, bonito, Candida albicans, Neurospora crassa, and spinach leaves. The results, which indicate that MSH production is restricted to the actinomycetes, could have significant implications for the detection and treatment of infections with actinomycetes, especially those caused by mycobacteria.

Organization of the Escherichia coli K-12 gene cluster responsible for production of the extracellular polysaccharide colanic acid.

Abstract: Colanic acid (CA) is an extracellular polysaccharide produced by most Escherichia coli strains as well as by other species of the family Enterobacteriaceae. We have determined the sequence of a 23-kb segment of the E. coli K-12 chromosome which includes the cluster of genes necessary for production of CA. The CA cluster comprises 19 genes. Two other sequenced genes (orf1.3 and galF), which are situated between the CA cluster and the O-antigen cluster, were shown to be unnecessary for CA production. The CA cluster includes genes for synthesis of GDP-L-fucose, one of the precursors of CA, and the gene for one of the enzymes in this pathway (GDP-D-mannose 4,6-dehydratase) was identified by biochemical assay. Six of the inferred proteins show sequence similarity to glycosyl transferases, and two others have sequence similarity to acetyl transferases. Another gene (wzx) is predicted to encode a protein with multiple transmembrane segments and may function in export of the CA repeat unit from the cytoplasm into the periplasm in a process analogous to O-unit export. The first three genes of the cluster are predicted to encode an outer membrane lipoprotein, a phosphatase, and an inner membrane protein with an ATP-binding domain. Since homologs of these genes are found in other extracellular polysaccharide gene clusters, they may have a common function, such as export of polysaccharide from the cell.

Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.

Abstract: One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated it close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.

PhoP-PhoQ activates transcription of pmrAB, encoding a two-component regulatory system involved in Salmonella typhimurium antimicrobial peptide resistance.

Abstract: Antimicrobial cationic peptides are a host defense mechanism of many animal species including mammals, insects, and amphibians. Salmonella typhimurium is an enteric and intracellular pathogen that interacts with antimicrobial peptides within neutrophil and macrophage phagosomes and at intestinal mucosal surfaces. The Salmonella spp. virulence regulators, PhoP and PhoQ, activate the transcription of genes (pag) within macrophage phagosomes necessary for resistance to cationic antimicrobial peptides. One PhoP-activated gene, pagB, forms an operon with pmrAB (5' pagB-pmrA-pmrB 3'), a two-component regulatory system involved in resistance to the antimicrobial peptides polymyxin, azurocidin (CAP37), bactericidal/permeability-increasing protein (BPI or CAP57), protamine, and polylysine. Expression of pmrAB increased transcription of pagB-pmrAB by activation of a promoter 5' to pagB. pmrAB is also expressed from a second promoter, not regulated by PhoP-PhoQ or PmrA-PmrB, located within the pagB coding sequence. S. typhimurium strains with increased pag locus expression were demonstrated to be polymyxin resistant because of induction of pagB-pmrAB; however, PmrA-PmrB was not responsible for the increased sensitivity of PhoP-null mutants to NP-1 defensin. Therefore, PhoP regulates at least two separate networks of genes responsible for cationic antimicrobial peptide resistance. These data suggest that resistance to the polymyxin-CAP family is controlled by a cascade of regulatory protein expression that activates transcription upon environmental sensing.

Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli.

Abstract: A study examining the influence of TolC on AcrA, AcrR, and MarR1 mutants indicates that functional TolC is required for the operation of the AcrAB efflux system and for the expression of the Mar phenotype. That the effect of TolC on the AcrAB pump is not regulatory in nature is shown by studies measuring the influence of a tolC::Tn10 insertion mutation on the expression of an acrA::lacZ reporter fusion. These results are compatible with the hypothesis that TolC is a component of the AcrAB efflux complex.

Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage.

Abstract: We have cloned the human mutY gene (hMYH) from both genomic and cDNA libraries. The human gene contains 15 introns and is 7.1 kb long. The 16 exons encode a protein of 535 amino acids that displays 41% identity to the Escherichia coli protein, which provides an important function in the repair of oxidative damage to DNA and helps to prevent mutations from oxidative lesions. The human mutY gene maps on the short arm of chromosome 1, between p32.1 and p34.3.

Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria.

Abstract: Evolutionary relationships among strictly anaerobic dissimilatory Fe(III)-reducing bacteria obtained from a diversity of sedimentary environments were examined by phylogenetic analysis of 16S rRNA gene sequences. Members of the genera Geobacter, Desulfuromonas, Pelobacter, and Desulfuromusa formed a monophyletic group within the delta subdivision of the class Proteobacteria. On the basis of their common ancestry and the shared ability to reduce Fe(III) and/or S0, we propose that this group be considered a single family, Geobacteraceae. Bootstrap analysis, characteristic nucleotides, and higher-order secondary structures support the division of Geobacteraceae into two subgroups, designated the Geobacter and Desulfuromonas clusters. The genus Desulfuromusa and Pelobacter acidigallici make up a distinct branch within the Desulfuromonas cluster. Several members of the family Geobacteraceae, none of which reduce sulfate, were found to contain the target sequences of probes that have been previously used to define the distribution of sulfate-reducing bacteria and sulfate-reducing bacterium-like microorganisms. The recent isolations of Fe(III)-reducing microorganisms distributed throughout the domain Bacteria suggest that development of 16S rRNA probes that would specifically target all Fe(III) reducers may not be feasible. However, all of the evidence suggests that if a 16S rRNA sequence falls within the family Geobacteraceae, then the organism has the capacity for Fe(III) reduction. The suggestion, based on geological evidence, that Fe(III) reduction was the first globally significant process for oxidizing organic matter back to carbon dioxide is consistent with the finding that acetate-oxidizing Fe(III) reducers are phylogenetically diverse.

The permeability of the wall fabric of Escherichia coli and Bacillus subtilis.

Abstract: To study the overall structure of the peptidoglycan fabric of the sacculi of gram-negative and gram-positive walls, actively growing cultures of Escherichia coli and Bacillus subtilis were treated with boiling sodium dodecyl sulfate solutions. The sacculi were then treated with enzymes to eliminate proteins and nucleic acids. These intact saccoli were probed with fluorescein-labeled dextrans with a range of known molecular weights. The penetration of the probes could be monitored by the negative-staining appearance in the fluorescence microscope. At several chosen times, the molecular weight fraction that allowed barely observable entry of the fluorescein-labeled probe and the molecular weight fraction that penetrated to achieve almost, but not quite, the concentration of probe in the solution external to the sacculi were determined. From three pairs of times and molecular weights that met one or the other of these two criteria, the effective pore size could be calculated. The minimum size of protein molecule that could diffuse through the pores was also calculated. Two mathematical models, which gave essentially the same results, were used to interpret the experimental data: one for the permeation of random coils through a surface containing holes and the other for rigid spheres diffusing through water-filled cylindrical pores. The mean estimate of the effective hole radius in walls from E. coli is 2.06 nm, and that of the effective hole size in walls from B. subtilis is 2.12 nm. These results are supported by experiments in which the loss of preloaded cells was monitored. Various fluorescein-labeled dextran samples were mixed with samples of intact cell walls, held for a long time, and then diluted. The efflux of the dextrans was monitored. Neither large nor small dextrans stained under these conditions. Only with dextran samples of a sufficiently small size were the sacculi filled during the preincubation period, and only with the largest of these could the probe not escape quickly. From the pore (or mesh) size, it can be concluded that the wall fabric of both organisms has few imperfections and that the major passageway is through the smallest possible pore, or "tessera," formed by the maximal cross-linking of the peptides from glycan chain to glycan chain compatible with the degree of rotational flexibility of the chains of repeating disaccharides of N-acetyl muramic acid and N-acetyl glucosamine. A tessera is composed of two chains of eight saccharides cross-linked by two octapeptides. The size of a globular hydrophilic molecule, if it did not bind to wall components, that could pass freely through the meshwork of an unstretched sacculus of either organism is roughly 25 kDa. We stress that this is only a rough estimate, and it may be possible for proteins of less than 50 kDa to pass through the native wall during normal growth conditions.

A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139.

Abstract: Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

Abstract: It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.

Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis.

Abstract: The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.

Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions.

Abstract: By a quantitative Western immunoblot analysis, the intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma S, the rpoS gene product), and of two minor sigma subunits, sigma 54 (sigma N, the rpoN gene product) and sigma 28 (sigma F, the rpoF gene product), were determined in two Escherichia coli strains, W3110 and MC4100. The results indicated that the levels of sigma 54 and sigma 28 are maintained at 10 and 50%, respectively, of the level of sigma 70 in both strains growing at both exponential and stationary phases, but in agreement with the previous measurement for strain MC4100 (M. Jishage and A. Ishihama, J. Bacteriol. 177:6832-6835, 1995), the level of sigma 38 was undetectable at the exponential growth phase but increased at 30% of the level of sigma 70 at the stationary phase. Stress-coupled change in the intracellular level was observed for two sigma subunits: (i) the increase in sigma 38 level and the decrease in sigma 28 level upon exposure to heat shock at the exponential phase and (ii) the increase in sigma 38 level under high-osmolality conditions at both the exponential and stationary phases.

SurA assists the folding of Escherichia coli outer membrane proteins.

Abstract: Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

Abstract: We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

Adhesion of the positively charged bacterium Stenotrophomonas (Xanthomonas) maltophilia 70401 to glass and Teflon.

Abstract: Medical implants are often colonized by bacteria which may cause severe infections. The initial step in the colonization, the adhesion of bacteria to the artificial solid surface, is governed mainly by long-range van der Waals and electrostatic interactions between the solid surface and the bacterial cell. While van der Waals forces are generally attractive, the usually negative charge of bacteria and solid surfaces leads to electrostatic repulsion. We report here on the adhesion of a clinical isolate, Stenotrophomonas maltophilia 70401, which is, at physiological pH, positively charged. S. maltophilia has an electrophoretic mobility of +0.3 x 10(-8) m2 V-1 s-1 at pH 7 and an overall surface isoelectric point at pH 11. The positive charge probably originates from proteins located in the outer membrane. For this bacterium, both long-range forces involved in adhesion are attractive. Consequently, adhesion of S. maltophilia to negatively charged surfaces such as glass and Teflon is much favored compared with the negatively charged bacterium Pseudomonas putida mt2. While adhesion of negatively charged bacteria is impeded in media of low ionic strength because of a thick negatively charged diffuse layer, adhesion of S. maltophilia was particularly favored in dilute medium. The adhesion efficiencies of S. maltophilia at various ionic strengths could be explained in terms of calculated long-range interaction energies between S. maltophilia and glass or Teflon.

Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease.

Abstract: In Escherichia coli, starvation (stationary-phase)-mediated differentiation involves 50 or more genes and is triggered by an increase in cellular sigma s levels. Western immunoblot analysis showed that in mutants lacking the protease ClpP or its cognate ATPase-containing subunit ClpX, sigma s levels of exponential-phase cells increased to those of stationary-phase wild-type cells. Lack of other potential partners of ClpP, i.e., ClpA or ClpB, or of Lon protease had no effect. In ClpXP-proficient cells, the stability of sigma s increased markedly in stationary-phase compared with exponential-phase cells, but in ClpP-deficient cells, sigma s became virtually completely stable in both phases. There was no decrease in ClpXP levels in stationary-phase wild-type cells. Thus, sigma s probably becomes more resistant to this protease in stationary phase. The reported sigma s-stabilizing effect of the hns mutation also was not due to decreased protease levels. Studies with translational fusions containing different lengths of sigma s coding region suggest that amino acid residues 173 to 188 of this sigma factor may directly or indirectly serve as at least part of the target for ClpXP protease.

Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11.

Abstract: Lactobacillus plantarum C11 secretes a small cationic peptide, plantaricin A, that serves as induction signal for bacteriocin production as well as transcription of plnABCD. The plnABCD operon encodes the plantaricin A precursor (PlnA) itself and determinants (PlnBCD) for a signal transducing pathway. By Northern (RNA) and sequencing analyses, four new plantaricin A-induced operons were identified. All were highly activated in concert with plnABCD upon bacteriocin induction. Two of these operons (termed plnEFI and plnJKLR) each encompass a gene pair (plnEF and plnJK, respectively) encoding two small cationic bacteriocin-like peptides with double-glycine-type leaders. The open reading frames (ORFs) encoding the bacteriocin-like peptides are followed by ORFs (plnI and -L, respectively) encoding cationic hydrophobic proteins resembling bacteriocin immunity proteins. On the third operon (termed plnMNOP), a similar bacteriocin-like ORF (plnN) and a putative immunity ORF (either plnM or -P) were identified as well. These findings suggest that two bacteriocins of two-peptide type (mature PlnEF and PlnJK) and a bacteriocin of one-peptide type (mature PlnN) could be responsible for the observed bacteriocin activity. The last operon (termed plnGHSTUV) contains two ORFs (plnGH) apparently encoding an ABC transporter and its accessory protein, respectively, known to be involved in processing and export of peptides with precursor double-glycine-type leaders. Promoter structure was established. A conserved regulatory-like box encompassing two direct repeats was identified in the promoter regions of all five plantaricin A-induced operons. These repeats may serve as regulatory elements for gene expression.

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

Abstract: The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.

Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes.

Abstract: The PhoP-PhoQ two-component system is essential for virulence in Salmonella typhimurium. This system controls expression of some 40 different proteins, yet most PhoP-regulated genes remain unknown. To identify PhoP-regulated genes, we isolated a library of 50,000 independent lac gene transcriptional fusion strains and investigated whether production of beta-galactosidase was regulated by PhoP. We recovered 47 lac gene fusions that were activated and 7 that were repressed when PhoP was expressed. Analysis of 40 such fusions defined some 30 loci, including mgtA and mgtCB, which encode two of the three Mg2+ uptake systems of S. typhimurium; ugd, encoding UDP-glucose dehydrogenase; phoP, indicative that the phoPQ operon is autoregulated; and an open reading frame encoding a protein with sequence similarity to VanX, a dipeptidase required for resistance to vancomycin. Transcription of PhoP-activated genes was regulated by the levels of Mg2+ in a PhoP-dependent manner. Strains with mutations in phoP or phoQ were defective for growth in low-Mg2+ media. The mgtA and mgtCB mutants reached lower optical densities than the wild-type strain in low-Mg2+ liquid media but displayed normal growth on low-Mg2+ solid media. Six PhoP-activated genes were identified as essential to form colonies on low-Mg'+ solid media. Cumulatively, our experiments establish that the PhoP-PhoQ system governs the adaptation to magnesium-limiting environments.

FtsZ ring formation in fts mutants.

Abstract: The formation of FtsZ rings (Z rings) in various fts mutants was examined by immunoelectron microscopy and immunofluorescence. In two temperature-sensitive ftsZ mutants which form filaments with smooth morphology, the Z ring was unable to form. In ftsA, ftsI, and ftsQ mutants, which form filaments with an indented morphology, Z rings formed but their contraction was blocked. These results indicate that fully functional ftsA, ftsQ, and ftsI genes are not required for Z-ring formation and are unlikely to have a role in localization of the Z ring. The results also suggest that one function of the Z ring is to localize the activity of other fts gene products.

Bacteriolytic effect of membrane vesicles from Pseudomonas aeruginosa on other bacteria including pathogens: conceptually new antibiotics.

Abstract: Pseudomonas aeruginosa releases membrane vesicles (MVs) filled with periplasmic components during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin. Natural and gentamicin-induced membrane vesicles (n-MVs and g-MVs, respectively) are subtly different from one another, but both contain several important virulence factors, including hydrolytic enzyme factors (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). Peptidoglycan hydrolases (autolysins) were detected in both MV types, especially a periplasmic 26-kDa autolysin whose expression has been related to growth phase (Z. Li, A. J. Clarke, and T. J. Beveridge, J. Bacteriol. 178:2479-2488, 1996). g-MVs possessed slightly higher autolysin activity and, at the same time, small quantities of gentamicin. Both MV types hydrolyzed isolated gram-positive and gram-negative murein sacculi and were also capable of hydrolyzing several glycyl peptides. Because the MVs were bilayered, they readily fused with the outer membrane of gram-negative bacteria. They also adhered to the cell wall of gram-positive bacteria. g-MVs were more effective in lysing other bacteria because, in addition to the autolysins, they also contained small amounts of gentamicin. The bactericidal activity was 2.5 times the MIC of gentamicin, which demonstrates the synergistic effect of the antibiotic with the autolysins. n-MVs were capable of killing cultures of P. aeruginosa with permeability resistance against gentamicin, indicating that the fusion of n-MV to the outer membrane liberated autolysins into the periplasm, where they degraded the peptidoglycan and lysed the cells. g-MVs had even greater killing power since they liberated both gentamicin and autolysins into these resistant cells. These findings may help develop a conceptually new group of antibiotics designed to be effective against hard-to-kill bacteria.