- Volume 143, Issue 8, 1997

Summary: Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.

Summary: The eicosapentaenoic acid (EPA) synthesis gene cluster isolated from a marine bacterium, Shewanella putrefaciens strain SCRC-2738, was cloned and expressed in the marine cyanobacterium Synechococcus sp. A broad-host-range cosmid vector, pJRD215 (10.2 kb, Smr Kmr), was used to clone a 38 kb insert, pEPA, containing the EPA synthesis gene cluster, creating plasmid pJRDEPA (approx. 48 kb). This plasmid was transferred to the cyanobacterial host at a frequency of 2.2 x 10−7. Cyanobacterial transconjugants grown at 29 °C produced 0.12 mg EPA (g dry weight)−1, whereas those grown at 23 °C produced 0.56 mg EPA (g dry weight)−1. The yield was further improved to 0.64 mg (g dry weight)−1 by incubation for 1 d at 17 °C. This is believed to be the first successful cloning and expression of such a large heterologous gene cluster in a marine cyanobacterium.

Summary: The physico-chemical cell-surface properties of Pseudomonas aeruginosa Al and its adhesion to silicone rubber under flow were compared for cells suspended in phosphate-buffered saline (PBS) or PBS supplemented with 2% nutrient broth. Addition of 2% nutrient broth to cells suspended in PBS yields minimal growth and did not significantly change the mean zeta potential of the organisms, which was around -13 mV. However, a comparatively larger proportion of the organisms had more negative zeta potentials in the presen of nutrient broth. This change was concurrent with a slight decrease in cell-surface hydrophobicity, as measured by water contact angles, from 119° to around 112°. The initial deposition rate of P. aeruginosa AK1 to silicone rubber, as studied in a parallel plate flow chamber, increased from 344 cm−2 s−1 in the absence of nutrient broth to 505 cm−2 s−1 in its presence. No stationary level of adhesion was observed in the presence of nutrient broth, instead the number of adhering cells increased steadily at a rate of approximately 85 cm−2 s−1. Fluorescent staining of adhering cells demonstrated that for adhesion from buffer only 2% of the adhering cells were metabolically active, whereas in case of deposition from PBS supplemented with nutrient broth, 67% of the adhering cells were metabolically active. It is concluded that the deposition rates measured in the parallel plate flow chamber with 2% nutrient broth added to the PBS suspension represent an interplay of adhesion and surface-associated growth.

Summary: Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using β-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity. bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungue, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J. T. (1995). Trends Biochem Sci 20, 178-179]. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CeIE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.

Summary: A secreted phosphate-repressible acid phosphatase from Kluyveromyces lactis has been purified and the N-terminal region and an internal peptide have been sequenced. Using synthetic oligodeoxyribonucleotides based on the sequenced regions, the genomic sequence, KIPHO5, encoding the protein has been isolated. The deduced protein, named KIPho5p, consists of 469 amino acids and has a molecular mass of 52 520 Da (in agreement with the data obtained after treatment of the protein with endoglycosidase H). The purified enzyme shows size heterogeneity, with an apparent molecular mass in the range 90-200 kDa due to the carbohydrate content (10 putative glycosylation sites were identified in the sequence). A 16 amino acid sequence at the N-terminus is similar to previously identified signal peptides in other fungal secretory proteins. The putative signal peptide is removed during secretion since it is absent in the mature secreted acid phosphatase. The gene can be induced 400-600-fold by phosphate starvation. Consensus signals corresponding to those described for Saccharomyces cerevisiae PHO4- and PHO2-binding sites are found in the 5′ region. Northern blot analysis of total cellular RNA indicates that the KIPHO5 gene codes for a 1.8 kb transcript and that its expression is regulated at the transcriptional level. Chromosomal hybridization indicated that the gene is located on chromosome II. The KIPHO5 gene of K. lactis is able to functionally complement a pho5 mutation of Sacch. cerevisiae. Southern blot experiments, using the KIPHO5 gene as probe, show that some K. lactis reference strains lack repressible acid phosphatase, revealing a different gene organization for this kind of multigene family of proteins as compared to Sacch. cerevisiae.

Summary: A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally held view of D29 as a virulent phage must be reviewed.

Summary: Four Lactobacillus strains (Lb. plantarum NCIMB 8826, Lb. paracasei LbTGS1.4, Lb. casei ATCC 393 and Lb. fermentum KLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an α-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest in Lb. plantarum NCIMB 8826, and lowest in Lb. paracasei LbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein from Streptococcus pyogenes fused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient in Lb. paracasei LbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereas Lb. plantarum NCIMB 8826 was able to secrete the antigen to a level as high as 10 mg I−1.

Summary: The DNA regions upstream from the genes encoding polypeptides of Bacillus thuringiensis subsp. israelensis larvicidal crystals (cry4A, cry4B, cry11A) contain sequences with similarities to the spoOA box of Bacillus subtilis (or ‘OA’ box) and the promoter recognized by the σH-associated RNA polymerase of B. subtilis. Expression of cry-lacZ transcriptional fusions was analysed in various B. thuringiensis genetic backgrounds. The early transcription of the toxin genes was not sporulation-dependent, whereas the late-stage expression at t 4-6 was σE-dependent. Primer extension analysis confirmed that the cry4-and cry11-type toxin genes were weakly transcribed during the transition phase; expression analysis of a cry11A'-lacZ transcriptional fusion in B. subtilis sporulation mutants confirmed the involvement of the σH-RNA polymerase. Primer extension analysis showed that in B. thuringiensis subsp. israelensis, the cry4A and cry11A gene transcription observed at the end of the growth stage was turned off at the beginning of the sporulation phase. The DNA region located upstream from the cry11A gene promoter including the putative ‘OA’ box was deleted. This led to a derepression of the expression of the cry11A operon. These results suggest that the cry4A, cry4B and cry11A toxin genes of B. thuringiensis subsp. israelensis are transcribed during the transition phase by the RNA polymerase associated with the σH factor and are subject to SpoOA repression.

Summary: Intercellular communication is a crucial phenomenon during spore development in Bacillus subtilis. It couples the establishment of a compartment-specific genetic program to the transcriptional activity of a σ factor in the other compartment. It also keeps σ factor activation in register with the morphological process. This study used directed mutagenesis to analyse the pathway that couples σE activation in the mother-cell to activation of σF in the forespore following asymmetric septation. Targets for mutagenesis in SpollGA (the receptor) were chosen based on the predicted topology of the protein when associated with the cell membrane. The results showed that a residue near the N terminus (D6), predicted to be exposed outside the cell, is required for receptor activity, whereas the major extracellular loop (between membrane domains IV and V) is dispensable for function. In contrast, mutations in SpollR (the signal) that partially blocked protein release (but not membrane translocation) had no effect on signal transduction. These results do not rule out the possibility that uncharacterized molecules intervene in the signalling pathway that establishes the mother-cell-specific developmental program during the early stage of sporulation.

Summary: Serratia marcescens is a nosocomial pathogen with a high incidence of β-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to β-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.

Summary: The replicator region of the symbiotic plasmid of Rhizobium etli CFN42 was cloned and sequenced. A plasmid derivative (pH3) harbouring a 5.6 kb HindIII fragment from the symbiotic plasmid was found to be capable of independent replication and eliminated the symbiotic plasmid when introduced into a R. etli CFNX101 strain (a recA derivative). The stability and the copy number of pH3 were the same as that of the symbiotic plasmid, indicating that the information required for stable replication and incompatibility resides in the 5.6 kb HindIII fragment. The sequence analysis of this fragment showed the presence of three ORFs similar in sequence and organization to repA, repB and repC described for the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b and for the R. leguminosarum cryptic plasmid pRL8JI. Hybridization studies showed that p42d-like replicator sequences are found in the symbiotic plasmids of other R. etli strains and in a ‘cryptic’ plasmid of R. tropici.

Summary: Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated XIn) which ranged in size from 2 kb to approximately 26 kb. Instability of DNA inserts in RP4::XIn hybrid plasmids was observed. The smallest insert present in a stable RP4::XIn6 hybrid plasmid, termed XIn6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA. Sequence analysis of XIn6 led to the discovery of an open reading frame with homology to the maturases of group II introns. The XIn6 insert also exhibited several features characteristic of a group II intron. These included the presence of the consensus sequence GUGYG at the 5′ end and RAY at the 3′ end of the intron. RNA secondary structure modelling of XIn6 also revealed the presence of perfectly conserved domains V and VI. Differences were detected in the XIn6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol. In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the XIn6 group II intron. This is the first reported group II intron isolated from Pseudomonas spp. and the first time that the mobility of a bacterial group II intron has been demonstrated.