Characterization of SepL of enterohemorrhagic Escherichia coli.

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions -5 and -36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37 degrees C, a nutrient-rich environment, high osmolarity, and the presence of Mn(2+). Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933DeltasepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.

Transcriptional regulation of the pas gene of enterohemorrhagic Escherichia coli.

The Pas protein plays a key role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC), being required for the secretion of the Esp proteins. Here, the transcriptional regulation of the pas gene was analyzed through the construction of a pas::lacZ translational fusion. When bacteria were grown in Luria Bertani medium or tissue culture medium supplemented with HEPES, a bimodal activation curve was observed. The early phase of induction was not significantly modified by the incubation temperature (either 25 or 37 degrees C), whereas the second phase, which overlaps with the late exponential growth phase, was enhanced at 37 degrees C. The early phase was also stimulated by growth on tissue culture medium and by the addition of Ca(2+), Mn(2+)or Mg(2+) to the M9-glucose minimal medium. Primer extension analysis showed the presence of two major starts of transcription, which were located 58 and 60 bp upstream of the ATG-start codon of the Pas protein, respectively. Although these sites are very close to each other, the transcripts produced during the early induction phase mainly start on the -60 position, whereas the -58 start was activated during the second induction phase.

Transcriptional regulation of the esp genes of enterohemorrhagic Escherichia coli.

We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemorrhagic Escherichia coli (EHEC) are organized in a single operon. The esp operon is controlled by a promoter located 94 bp upstream from the ATG start codon of the espA gene. The promoter is activated in the early logarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca2+, Mn2+, and HEPES. Transcription of the esp operon seems to be switched off in tightly attached bacteria. The activation process is regulated by osmolarity (induction at high osmolarities), modulated by temperature, and influenced by the degree of DNA supercoiling. Transcription is sigmaS dependent, and the H-NS protein contributes to its fine tuning. Identification of the factors involved in activation of the esp operon and the signals responsible for modulation may facilitate understanding of the underlying molecular events leading to sequential expression of virulence factors during natural infections caused by EHEC.

Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST.

The nucleotide sequence of the 4,377-bp chromosomal region of Pseudomonas fluorescens ST that codes for the oxidation of styrene to phenylacetic acid was determined. Four open reading frames, named styA, styB, styC, and styD, were identified in this region. Sequence analysis and biotransformation assays, performed with batch and continuous cultures, allowed us to identify the functions of the sequenced genes. styA and styB encode a styrene monooxygenase responsible for the transformation of styrene to epoxystyrene; styC codes for the second enzyme of the pathway, an epoxystyrene isomerase that converts epoxystyrene to phenylacetaldehyde; and the styD gene produces a phenylacetaldehyde dehydrogenase that oxidizes phenylacetaldehyde to phenylacetic acid. StyA, 415-amino-acids long, was found to be weakly homologous to p-hydroxybenzoate hydroxylase from both P. fluorescens and P. aeruginosa and to salicylate hydroxylase from P. putida, suggesting that it might be a flavin adenine dinucleotide-binding monooxygenase. StyB was found to be partially homologous to the carboxyterminal part of the 2,4-dichlorophenol-6-monooxygenase encoded by plasmid pJP4, while the styC product did not share significant homology with any known proteins. The fourth open reading frame, styD, could encode a protein of 502 amino acids and was strongly homologous to several eukaryotic and prokaryotic aldehyde dehydrogenases. The order of the genes corresponds to that of the catabolic steps. The previously suggested presence of the gene for epoxystyrene reductase, which directly converts epoxystyrene to 2-phenylethanol (A.M. Marconi, F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro, Appl. Environ. Microbiol. 61:121-127, 1996), has not been confirmed by sequencing and by biotransformation assays performed in continuous cultures. A copy of the insertion sequence ISI162, belonging to the IS21-like family of elements, was identified immediately downstream of the styrene catabolic genes.

Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST.

A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.