Journal of Bacteriology

Harris, Baruch Z.; Singer, Mitchell

doi: N/Apmid: 9829957

The chromosomal acetylornithine deacetylase ( argE ) gene of Myxococcus xanthus was identified via homology to acetylornithine deacetylases from other bacterial species. A mutant carrying a disruption in argE was unable to grow on minimal media lacking supplemental arginine and formed fruiting bodies and spores in response to arginine starvation at high cell density.

Bai, Qing; Somerville, Ronald L.

doi: N/Apmid: 9829925

The tpl gene of Citrobacter freundii encodes an enzyme that catalyzes the conversion of L -tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates 272.5, 158.5, and 49.5, respectively, were identified in the upstream region of the tpl promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of tpl required integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate 85 and that there are CRP binding sites centered at coordinates 220 and 250. Mutational alteration of the IHF binding site reduced the efficiency of the tpl promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into tpl promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased when tpl DNA was present. The participation of both IHF and CRP in the activation of the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of the tpl promoter is presented.

Xu, Di; Yang, Chun; Kaplan, Heidi B.

doi: N/Apmid: 9829930

Myxococcus xanthus multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations ( sasB5 , - 14 , - 15 , - 16 , and - 17 ) can bypass the starvation and high-cell-density requirements for expression of the 4521 developmental reporter gene. These mutants express 4521 at high levels during growth and development in an asgB background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the sasB locus cloned from the wild-type chromosome restored the SasB + phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed sasN -null mutation and 4521 Tn 5lac in an otherwise wild-type background expressed 4521 at a high level during growth and development. A similar sasN -null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type sasN gene product is necessary for normal M. xanthus fruiting body development and functions as a critical regulator that prevents 4521 expression during growth.

Belas, Robert; Schneider, Rachel; Melch, Michael

doi: N/Apmid: 9829920

Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull's-eye colony often associated with cultures of P. mirabilis . How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA . Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.

Buddelmeijer, Nienke; Aarsman, Mirjam E. G.; Kolk, Arend H. J.; Vicente, Miguel; Nanninga, Nanne

doi: N/Apmid: 9829918

The localization of cell division protein FtsQ in Escherichia coli wild-type cells was studied by immunofluorescence microscopy with specific monoclonal antibodies. FtsQ could be localized to the division site in constricting cells. FtsQ could also localize to the division site in ftsQ1 (Ts) cells grown at the permissive temperature. A hybrid protein in which the cytoplasmic domain and the transmembrane domain were derived from the form of penicillin-binding protein 1B and the periplasmic domain was derived from FtsQ was also able to localize to the division site. This result indicates that the periplasmic domain of FtsQ determines the localization of FtsQ, as has also been concluded by others for the periplasmic domain of FtsN. Noncentral FtsQ foci were found in the area of the cell where the nucleoid resides and were therefore assumed to represent sites where the FtsQ protein is synthesized and simultaneously inserted into the cytoplasmic membrane.

Belitsky, Boris R.; Sonenshein, Abraham L.

doi: N/Apmid: 9829940

Role and Regulation of Bacillus subtilis Glutamate Dehydrogenase Genes Boris R. Belitsky * and Abraham L. Sonenshein Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 ABSTRACT The complete Bacillus subtilis genome contains two genes with the potential to encode glutamate dehydrogenase (GlutDH) enzymes. Mutations in these genes were constructed and characterized. The rocG gene proved to encode a major GlutDH whose synthesis was induced in media containing arginine or ornithine or, to a lesser degree, proline and was repressed by glucose. A rocG null mutant was impaired in utilization of arginine, ornithine, and proline as nitrogen or carbon sources. The gudB gene was expressed under all growth conditions tested but codes for a GlutDH that seemed to be intrinsically inactive. Spontaneous mutations in gudB that removed a 9-bp direct repeat within the wild-type gudB sequence activated the GudB protein and allowed more-efficient utilization of amino acids of the glutamate family.

Chan, Pan F.; Foster, Simon J.

doi: N/Apmid: 9829932

Role of SarA in Virulence Determinant Production and Environmental Signal Transduction in Staphylococcus aureus Pan F. Chan and Simon J. Foster * Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom ABSTRACT The staphylococcal accessory regulator (encoded by sarA ) is an important global regulator of virulence factor biosynthesis in Staphylococcus aureus . To further characterize its role in virulence determinant production, an sarA knockout mutant was created by insertion of a kanamycin antibiotic resistance cassette into the sarA gene. N-terminal sequencing of exoproteins down-regulated by sarA identified several putative proteases, including a V8 serine protease and a novel metalloprotease, as the major extracellular proteins repressed by sarA . In kinetic studies, the sarA mutation delays the onset of α-hemolysin (encoded by hla ) expression and reduces levels of hla to approximately 40% of the parent strain level. Furthermore, SarA plays a role in signal transduction in response to microaerobic growth since levels of hla were much lower in a microaerobic environment than after aerobic growth in the sarA mutant. An exoprotein exhibiting hemolysin activity on sheep blood, and up-regulated by sarA independently of the accessory gene regulator (encoded by agr ), was specifically induced microaerobically. Transcriptional gene fusion and Western analysis revealed that sarA up-regulates both toxic shock syndrome toxin 1 gene ( tst ) expression and staphylococcal enterotoxin B production, respectively. This study demonstrates the role of sarA as a signal transduction regulatory component in response to aeration stimuli and suggests that sarA functions as a major repressor of protease activity. The possible role of proteases as regulators of virulence determinant stability is discussed.

Tapias, Angels; Barbe, Jordi

doi: N/Apmid: 9829943

Based upon our earlier studies (A. Tapias, A. R. Fernández de Henestrosa, and J. Barbé, J. Bacteriol. 179:1573-1579, 1997) we hypothesized that the regulatory sequence of the Rhizobium etli recA gene was TTGN 11 CAA. However, further detailed analysis of the R. etli recA operator described in the present work suggests that it may in fact be GAACN 7 GTAC. This new conclusion is based upon PCR mutagenesis analysis carried out in the R. etli recA operator, which indicates that the GAAC and GTAC submotifs found in the sequence GAACN 7 GTAC are required for the maximal stimulation of in vivo transcription and in vitro DNA-protein complex formation. This DNA-protein complex is also detected when the GAACN 7 GTAC wild-type sequence is modified to obtain GAACN 7 GAAC, GTACN 7 GTAC, or GAACN 7 GTTC. The wild-type promoters of the Rhizobium meliloti and Agrobacterium tumefaciens recA genes, which also contain the GAACN 7 GTAC sequence, compete with the R. etli recA promoter for the DNA-protein complex formation but not with mutant derivatives in any of these motifs, indicating that the R. etli , R. meliloti , and A. tumefaciens recA genes present the same regulatory sequence.

Fischer, Daniela; Teich, Antje; Neubauer, Peter; Hengge-Aronis, Regine

doi: N/Apmid: 9829928

The general stress sigma factor S (RpoS) of Escherichia coli is strongly induced in response to glucose starvation. This increase in the cellular S level is due to stabilization of S , which under non-stress conditions is subject to rapid proteolysis. In the present study, it is demonstrated that S is also induced during the diauxic shift from glucose to lactose, i.e., under conditions of glucose exhaustion in the presence of another, less-preferred carbon source that eventually gets utilized. This S induction, which is due to stabilization, is transient and precedes the induction of -galactosidase. In parallel, S -dependent genes are transiently activated, as was shown here for osmY . Although S can mediate transcription of lacZ in vitro, S does not contribute to the induction of -galactosidase during the diauxic lag phase. Rather, the induction of S and the general stress response during the diauxic shift plays the role of a rapidly activated emergency system, which is shut off again as soon as the cells are able to cope with the stress situation by utilizing a more specific and more economical system.

Johansson, Jörgen; Dagberg, Björn; Richet, Evelyne; Uhlin, Bernt Eric

doi: N/Apmid: 9829919

H-NS and StpA Proteins Stimulate Expression of the Maltose Regulon in Escherichia coli Jörgen Johansson 1 , Björn Dagberg 2 , Evelyne Richet 3 , and Bernt Eric Uhlin 1 , * Department of Microbiology 1 and Department of Physiology, 2 Umeå University, S-90187 Umeå, Sweden, and Unité de Génétique Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France 3 ABSTRACT The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in Escherichia coli . Its role in gene regulation is manifested by the increased expression of several gene products in hns mutant strains. Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon. In studies with hns mutant strains and derivatives also deficient in the stpA gene, we found that expression of the LamB porin was decreased. Our results showed that the amounts of both LamB protein and lamB mRNA were greatly reduced in hns and hns-stpA mutant strains. The same results were obtained when we monitored the amount of transcription from the malEFG operon. The lamB gene is situated in the malKlamBmalM operon, which forms a divergent operon complex together with the malEFG operon. The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein. Using a malT-lacZ translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in hns and hns-stpA mutant strains in comparison with the wild-type strain. Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon.