Journal of Bacteriology

Kristoffersen, Simen M.; Ravnum, Solveig; Tourasse, Nicolas J.; Okstad, Ole Andreas; Kolsto, Anne-Brit; Davies, William

doi: 10.1128/JB.00239-07pmid: 17496091

Tolerance to bile salts was investigated in forty Bacillus cereus strains, including 17 environmental isolates, 11 dairy isolates, 3 isolates from food poisoning outbreaks, and 9 other clinical isolates. Growth of all strains was observed at low bile salt concentrations, but no growth was observed on LB agar plates containing more than 0.005% bile salts. Preincubation of the B. cereus type strain, ATCC 14579, in low levels of bile salts did not increase tolerance levels. B. cereus ATCC 14579 was grown to mid-exponential growth phase and shifted to medium containing bile salts (0.005%). Global expression patterns were determined by hybridization of total cDNA to a 70-mer oligonucleotide microarray. A general stress response and a specific response to bile salts were observed. The general response was similar to that observed in cultures grown in the absence of bile salts but at a higher (twofold) cell density. Up-regulation of several putative multidrug exporters and transcriptional regulators and down-regulation of most motility genes were observed as part of the specific response. Motility experiments in soft agar showed that motility decreased following bile salts exposure, in accordance with the transcriptional data. Genes encoding putative virulence factors were either unaffected or down-regulated.

Murray, Sean R.; Ernst, Robert K.; Bermudes, David; Miller, Samuel I.; Low, K. Brooks

doi: 10.1128/JB.01969-06pmid: 17449614

Mutations in pmrA were recombined into Salmonella strain ATCC 14028 msbB to determine if pmrA -regulated modifications of lipopolysaccharide could suppress msbB growth defects. A mutation that functions to constitutively activate pmrA pmrA (Con) suppresses msbB growth defects on EGTA-containing media. Lipid A structural analysis showed that Salmonella msbB pmrA (Con) strains, compared to Salmonella msbB strains, have increased amounts of palmitate and phosphoethanolamine but no aminoarabinose addition, suggesting that aminoarabinose is not incorporated into msbB lipid A. Surprisingly, loss-of-function mutations in the aminoarabinose biosynthetic genes restored EGTA and polymyxin sensitivity to Salmonella msbB pmrA (Con) strains. These blocks in aminoarabinose biosynthesis also prevented lipid A phosphoethanolamine incorporation and reduced the levels of palmitate addition, indicating previously unknown roles for the aminoarabinose biosynthetic enzymes. Lipid A structural analysis of the EGTA- and polymyxin-resistant triple mutant msbB pmrA (Con) pagP ::Tn 10 , which contains phosphoethanolamine but no palmitoylated lipid A, suggests that phosphoethanolamine addition is sufficient to confer EGTA and polymyxin resistance on Salmonella msbB strains. Additionally, palmitoylated lipid A was observed only in wild-type Salmonella grown in the presence of salt in rich media. Thus, we correlate EGTA resistance and polymyxin resistance with phosphoethanolamine-decorated lipid A and demonstrate that the aminoarabinose biosynthetic proteins play an essential role in lipid A phosphoethanolamine addition and affect lipid A palmitate addition in Salmonella msbB strains.

Priyadarshini, Richa; de Pedro, Miguel A.; Young, Kevin D.

doi: 10.1128/JB.00415-07pmid: 17483214

Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.

del Castillo, Teresa; Ramos, Juan L.; Rodriguez-Herva, Jose J.; Fuhrer, Tobias; Sauer, Uwe; Duque, Estrella

doi: 10.1128/JB.00203-07pmid: 17483213

In this study, we show that glucose catabolism in Pseudomonas putida occurs through the simultaneous operation of three pathways that converge at the level of 6-phosphogluconate, which is metabolized by the Edd and Eda Entner/Doudoroff enzymes to central metabolites. When glucose enters the periplasmic space through specific OprB porins, it can either be internalized into the cytoplasm or be oxidized to gluconate. Glucose is transported to the cytoplasm in a process mediated by an ABC uptake system encoded by open reading frames PP1015 to PP1018 and is then phosphorylated by glucokinase (encoded by the glk gene) and converted by glucose-6-phosphate dehydrogenase (encoded by the zwf genes) to 6-phosphogluconate. Gluconate in the periplasm can be transported into the cytoplasm and subsequently phosphorylated by gluconokinase to 6-phosphogluconate or oxidized to 2-ketogluconate, which is transported to the cytoplasm, and subsequently phosphorylated and reduced to 6-phosphogluconate. In the wild-type strain, glucose was consumed at a rate of around 6 mmol g –1 h –1 , which allowed a growth rate of 0.58 h –1 and a biomass yield of 0.44 g/g carbon used. Flux analysis of 13 C-labeled glucose revealed that, in the Krebs cycle, most of the oxalacetate fraction was produced by the pyruvate shunt rather than by the direct oxidation of malate by malate dehydrogenase. Enzymatic and microarray assays revealed that the enzymes, regulators, and transport systems of the three peripheral glucose pathways were induced in response to glucose in the outer medium. We generated a series of isogenic mutants in one or more of the steps of all three pathways and found that, although all three functioned simultaneously, the glucokinase pathway and the 2-ketogluconate loop were quantitatively more important than the direct phosphorylation of gluconate. In physical terms, glucose catabolism genes were organized in a series of clusters scattered along the chromosome. Within each of the clusters, genes encoding porins, transporters, enzymes, and regulators formed operons, suggesting that genes in each cluster coevolved. The glk gene encoding glucokinase was located in an operon with the edd gene, whereas the zwf-1 gene, encoding glucose-6-phosphate dehydrogenase, formed an operon with the eda gene. Therefore, the enzymes of the glucokinase pathway and those of the Entner-Doudoroff pathway are physically linked and induced simultaneously. It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with glucose.

Pilonieta, M. Carolina; Bodero, Maria D.; Munson, George P.

doi: 10.1128/JB.00131-07pmid: 17496090

H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp . One site extends from positions –23 to –56, and the other extends from positions –73 to –103 (numbering relative to the transcription start site of cfaAp ). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE . In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp :: lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

Duerkop, Breck A.; Ulrich, Ricky L.; Greenberg, E. Peter

doi: 10.1128/JB.00317-07pmid: 17496085

Acyl-homoserine lactones (HSLs) serve as quorum-sensing signals for many Proteobacteria . Members of the LuxI family of signal generators catalyze the production of acyl-HSLs, which bind to a cognate receptor in the LuxR family of transcription factors. The obligate animal pathogen Burkholderia mallei produces several acyl-HSLs, and the B. mallei genome has four luxR and two luxI homologs, each of which has been established as a virulence factor. To begin to delineate the relevant acyl-HSL signals for B. mallei LuxR homologs, we analyzed the BmaR1-BmaI1 system. A comparison of acyl-HSL profiles from B. mallei ATCC 23344 and a B. mallei bmaI1 mutant indicates that octanoyl-HSL synthesis is BmaI1 dependent. Furthermore, octanoyl-HSL is the predominant acyl-HSL produced by BmaI1 in recombinant Escherichia coli . The synthesis of soluble BmaR1 in recombinant E. coli requires octanoyl-HSL or decanoyl-HSL. Insoluble aggregates of BmaR1 are produced in the presence of other acyl-HSLs and in the absence of acyl-HSLs. The bmaI1 promoter is activated by BmaR1 and octanoyl-HSL, and a 20-bp inverted repeat in the bmaI1 promoter is required for bmaI1 activation. Purified BmaR1 binds to this promoter region. These findings implicate octanoyl-HSL as the signal for BmaR1-BmaI1 quorum sensing and show that octanoyl-HSL and BmaR1 activate bmaI1 transcription.

Ballicora, Miguel A.; Erben, Esteban D.; Yazaki, Terutaka; Bertolo, Ana L.; Demonte, Ana M.; Schmidt, Jennifer R.; Aleanzi, Mabel; Bejar, Clarisa M.; Figueroa, Carlos M.; Fusari, Corina M.; Iglesias, Alberto A.; Preiss, Jack

doi: 10.1128/JB.00481-07pmid: 17496097

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu 102 and Pro 103 was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.

Byrne, Gavin A.; Russell, Dean A.; Chen, Xiaoxiao; Meijer, Wim G.

doi: 10.1128/JB.00431-07pmid: 17496078

The virR operon, located on the virulence plasmid of the intracellular pathogen Rhodococcus equi , contains five genes, two of which ( virR and orf8 ) encode transcriptional regulators. The first gene of the operon ( virR ), encoding a LysR-type transcriptional regulator, is transcribed at a constitutive low level, whereas the four downstream genes are induced by low pH and high growth temperature. Differential regulation of the virR operon genes could not be explained by differential mRNA stability, as there were no major differences in mRNA half-lives of the transcripts representing each of the five genes within the virR operon. Transcription of virR is driven by the P virR promoter, with a transcription start site 53 bp upstream of the virR initiation codon. The four genes downstream of virR are transcribed from P virR and from a second promoter, P orf5 , located 585 bp downstream of the virR initiation codon. VirR binds to a site overlapping the initiation codon of virR , resulting in negative autoregulation of the virR gene, explaining its low constitutive transcription level. The P orf5 promoter is induced by high temperature and low pH, thus explaining the observed differential gene expression of the virR operon. VirR has a positive effect on P orf5 activity, whereas the response regulator encoded by orf8 is not involved in regulating transcription of the virR operon. The P virR promoter is strikingly similar to those recognized by the principal sigma factors of Streptomyces and Mycobacterium , whereas the P orf5 promoter does not share sequence similarity with P virR . This suggests that P orf5 is recognized by an alternative sigma factor.

Lee, Yvonne M.; Dodson, Karen W.; Hultgren, Scott J.

doi: 10.1128/JB.01648-06pmid: 17496084

P-pilus biogenesis occurs via the highly conserved chaperone-usher pathway and involves the strict coordination of multiple subunit proteins. All nonadhesin structural P-pilus subunits possess the same topology, consisting of two domains: an incomplete immunoglobulin-like fold (pilin body) and an N-terminal extension. Pilus subunits form interactions with one another through donor strand exchange, occurring at the usher, in which the N-terminal extension of an incoming subunit completes the pilin body of the preceding subunit, allowing the incorporation of the subunit into the pilus fiber. In this study, pilus subunits in which the N-terminal extension was either deleted or swapped with that of another subunit were used to examine the role of each domain of PapF in functions involving donor strand exchange and hierarchical assembly. We found that the N-terminal extension of PapF is required to adapt the PapG adhesin to the tip of the fiber. The pilin body of PapF is required to efficiently initiate assembly of the remainder of the pilus, with the assistance of the N-terminal extension. Thus, distinct functions were assigned to each region of the PapF subunit. In conclusion, all pilin subunits possess the same overall architectural topology; however, each N-terminal extension and pilin body has specific functions in pilus biogenesis.

Sakuragi, Yumiko; Kolter, Roberto

doi: 10.1128/JB.00137-07pmid: 17496081

Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.