Journal of Bacteriology

Chernin, Leonid S.; Winson, Michael K.; Thompson, Jacquelyn M.; Haran, Shoshan; Bycroft, Barrie W.; Chet, Ilan; Williams, Paul; Stewart, Gordon S. A. B.

doi: N/Apmid: 9721280

Quorum sensing control mediated by N -acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In Chromobacterium violaceum ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL N -hexanoyl- L -homoserine lactone (HHL). In this study we show that C. violaceum produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using p -nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of N -acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of N -acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two N -acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn 5 mutant of C. violaceum (CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the C. violaceum wild-type strain or 10 µM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.

Qian, Yilei; Tabita, F. Robert

doi: N/Apmid: 9721307

Expression of glnB and a glnB -Like Gene ( glnK ) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides Yilei Qian 1 and F. Robert Tabita 1 , 2 , * The Biochemistry Program 1 and The Department of Microbiology and Plant Molecular Biology/Biotechnology Program, 2 The Ohio State University, Columbus, Ohio 43210-1292 ABSTRACT In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides , strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB :: lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB -like gene, glnK , was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.

Tal, Rony; Wong, Hing C.; Calhoon, Roger; Gelfand, David; Fear, Anna Lisa; Volman, Gail; Mayer, Raphael; Ross, Peter; Amikam, Dorit; Weinhouse, Haim; Cohen, Avital; Sapir, Shai; Ohana, Patricia; Benziman, Moshe

doi: N/Apmid: 9721278

Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of -1,4-glucan (cellulose) synthase in Acetobacter xylinum . The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1 , cdg2 , and cdg3 . Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.

Helling, Robert B.

doi: N/Apmid: 9721297

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog -methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.

Garza, Anthony G.; Pollack, Jeffrey S.; Harris, Baruch Z.; Lee, Albert; Keseler, Ingrid M.; Licking, Ellen F.; Singer, Mitchell

doi: N/Apmid: 9721305

Myxococcus xanthus cells carrying the 4408 Tn 5lac insertion at the sde locus show defects in fruiting body development and sporulation. Our analysis of sde expression patterns showed that this locus is induced early in the developmental program (0 to 2 h) and that expression increases approximately fivefold after 12 h of development. Further studies showed that expression of sde is induced as growing cells enter stationary phase, suggesting that activation of the sde locus is not limited to the developmental process. Because the peak levels of sde expression in both an sde + and an sde mutant background were similar, we conclude that the sde locus is not autoregulated. Characterization of the sde locus by DNA sequence analysis indicated that the 4408 insertion occurred within the sdeK gene. Primer extension analyses localized the 5' end of sde transcript to a guanine nucleotide 307 bp upstream of the proposed start for the SdeK coding sequence. The DNA sequence in the 12 and 24 regions upstream of the sde transcriptional start site shows similarity to the 54 family of promoters. The results of complementation studies suggest that the defects in development and sporulation caused by the 4408 insertion are due to an inactivation of sdeK . The predicted amino acid sequence of SdeK was found to have similarity to the sequences of the histidine protein kinases of two-component regulatory systems. Based on our results, we propose that SdeK may be part of a signal transduction pathway required for the activation and propagation of the early developmental program.

Mhlanga-Mutangadura, Tendai; Morlin, Gregory; Smith, Arnold L.; Eisenstark, Abraham; Golomb, Miriam

doi: N/Apmid: 9721313

Haemophilus influenzae is a ubiquitous colonizer of the human respiratory tract and causes diseases ranging from otitis media to meningitis. Many H. influenzae isolates express pili (fimbriae), which mediate adherence to epithelial cells and facilitate colonization. The pilus gene ( hif ) cluster of H. influenzae type b maps between purE and pepN and resembles a pathogenicity island: it is present in invasive strains, absent from the nonpathogenic Rd strain, and flanked by direct repeats of sequence at the insertion site. To investigate the evolution and role in pathogenesis of the hif cluster, we compared the purE-pepN regions of various H. influenzae laboratory strains and clinical isolates. Unlike Rd, most strains had an insert at this site, which usually was the only chromosomal locus of hif DNA. The inserts are diverse in length and organization: among 20 strains, nine different arrangements were found. Several nontypeable isolates lack hif genes but have two conserved open reading frames ( hicA and hicB ) upstream of purE ; their inferred products are small proteins with no data bank homologs. Other isolates have hif genes but lack hic DNA or have combinations of hif and hic genes. By comparing these arrangements, we have reconstructed a hypothetical ancestral genotype, the extended hif cluster. The hif region of INT1, an invasive nontypeable isolate, resembles the hypothetical ancestor. We propose that a progenitor strain acquired the extended cluster by horizontal transfer and that other variants arose as deletions. The structure of the hif cluster may correlate with colonization site or pathogenicity.

Nikaido, Hiroshi; Basina, Marina; Nguyen, Vy; Rosenberg, Emiko Y.

doi: N/Apmid: 9721312

We found that the previously reported SS-B drug-supersusceptible mutant of Salmonella typhimurium (S. Sukupolvi, M. Vaara, I. M. Helander, P. Viljanen, and P. H. Mäkelä, J. Bacteriol. 159:704-712, 1984) had a mutation in the acrAB operon. Comparison of this mutant with its parent strain and with an AcrAB-overproducing strain showed that the activity of the AcrAB efflux pump often produced significant resistance to -lactam antibiotics in the complete absence of -lactamase. The effect of AcrAB activity on resistance was more pronounced with agents containing more lipophilic side chains, suggesting that such compounds were better substrates for this pump. This correlation is consistent with the hypothesis that only those molecules that become at least partially partitioned into the lipid bilayer of the cytoplasmic membrane are captured by the AcrAB pump. According to this mechanism, the pump successfully excretes even those -lactams that fail to traverse the cytoplasmic membrane, because these compounds are likely to become partitioned into the outer leaflet of the bilayer. Even the compounds with lipophilic side chains were shown to penetrate across the outer membrane relatively rapidly, if the pump was inactivated genetically or physiologically. The exclusion of such compounds, exemplified by nafcillin, from cells of the wild-type S. typhimurium was previously interpreted as the result of poor diffusion across the outer membrane (H. Nikaido, Biochim. Biophys. Acta 433:118-132, 1976), but it is now recognized as the consequence of efficient pumping out of entering antibiotics by the active efflux process.

McLenigan, Mary; Peat, Thomas S.; Frank, Ekaterina G.; McDonald, John P.; Gonzalez, Martin; Levine, Arthur S.; Hendrickson, Wayne A.; Woodgate, Roger

doi: N/Apmid: 9721309

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD', the function of UmuD' has yet to be determined. In an attempt to elucidate the role of UmuD' in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD ' plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD ' mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD', three were nonsense mutations that resulted in a truncated UmuD' protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1 , umuD44 , and umuD77 alleles into umuD '. All 17 umuD ' mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD'.

Horiuchi, Takayuki; Komano, Teruya

doi: N/Apmid: 9721303

The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the pilS product, and a minor component, one of the seven pilV products. The pilS product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of the pilU product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with the pilV product. To reveal the relationship between the structure and function of the pilS product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant pilS products were analyzed. Four pilS mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type pilS gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the pilS product requires the entire prepilin sequence.

Torgov, Michael Y.; Janzen, Deanna M.; Reddy, Michael K.

doi: N/Apmid: 9721267

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the "sliding clamp" of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the "clamp loader" activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes 44 and 62 , which are directly adjacent on polycistronic mRNA molecules, are to some degree translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene 44 . Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of ~85% and a coupling frequency of ~25% between the 44-62 gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.