- Volume 141, Issue 10, 1995

Summary: Five of the genes known to encode the enzymes for the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons, tagAB and tagDEF. lacZ and gus transcriptional fusions to the first genes of these operons revealed that: (i) in media of different richness, higher growth rates were paralleled by lower transcription levels; (ii) upon transition to stationary phase, the transcription per unit mass of both operons increased abruptly by a factor of about two; and (iii) a rise in temperature was accompanied by decreased transcription of tagA and increased transcription of tagD. Mapping of transcription start points revealed two divergent aA-controlled promoters. Although tagD and the neighbouring downstream gene tagE are transcribed from the same promoter, the latter was expressed at a much lower level than the former. Moreover, expression of tagE, and of the translationally coupled tagF, did not increase at the onset of the stationary phase, indicating that additional regulatory signals may act in the intergenic tagD-tagE region. Optimal transcription of these operons appears to require the entire regulatory region, suggesting that tag gene expression may, among other factors, be regulated by the three-dimensional configuration of this segment. The biological implications of these results are discussed.

Summary: The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = cwlG) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a lytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase. The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The lytD gene product, in either an intact or a truncated form, was found to be lethal for E. coli, and the N-terminally truncated glucosaminidase proteins, produced in E. coli, were very unstable. The partially purified glucosaminidase from B. subtilis was found to be very unstable at low ionic strength at 37 °C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.

Summary: The tbpA and tbpB genes encoding the transferrin receptor proteins Tbp 1 and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB gene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic sequence suggesting that the two genes may be coordinately transcribed. Determination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparative purposes. The deduced amino acid sequences of the Tbp1 proteins had regions of homology with Neisseria Lbp and Tbp1s and with TonB-dependent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presented in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant Tbp1 was found in inclusion bodies and could not be affinity-isolated with immobilized porcine transferrin. Most of the remaining expressed Tbp1 was present in the OM fraction, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the C-lobe of porcine transferrin. Although recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM fraction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbp1. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transferrin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the C-lobe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins have similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with recombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.

Summary: Two site-specific DNA rearrangements occur during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120: the deletion of an 11 kb element from within the nifD gene and the deletion of a 55 kb element from within the fdxN gene. Three Nostoc and six Anabaena strains were screened for the presence of the nifD and fdxN elements by Southern hybridization with Anabaena PCC 7120 DNA probes. Eight of the nine strains contained DNA sequences that were similar to the nifD element. Three strains, Nostoc sp. strain Mac, Anabaena cylindrica and Anabaena sp. strain M131, also showed significant similarity to portions of the 55 kb fdxN element. Anabaena sp. strain CA lacked both the nifD and fdxN elements. Southern analysis of vegetative cell and heterocyst DNA from A. cylindrica and a Fox+ revertant of Nostoc Mac (isolate R2) showed rearrangement of the nifD and fdxN elements in heterocysts. We found no RFLPs between Anabaena M131 and Anabaena PCC 7120 suggesting that strain M131 is a Het- derivative of strain PCC 7120.

Summary: One of the critical developmental events during the unique intracellular life cycle of Chlamydiae is their differentiation from a metabolically active, replicative form or reticulate body (RB) to an infectious extracellular form of the organism (elementary body or EB). This process is characterized by the expression of two extraordinarily cysteine-rich envelope proteins of molecular masses 9 kDa and 60 kDa. We describe the molecular cloning and sequence determination of the 9 kDa cysteine-rich proteins (CrPs) of C. pneumoniae and C. psittaci. Comparison of these 9 kDa CrP amino acid sequences with those of C. trachomatis showed regions of structural variation and conservation. Transcription of the 9 kDa CrP genes occurred as both a monocistronic message and as a bicistronic message which included the 60 kDa CrP gene. Transcription of the 9 kDa and 60 kDa CrP genes was tightly linked to the chlamydial growth cycle with synthesis of their mRNAs and consequent translation of the 60 kDa CrPs occurring as RBs differentiated to form EBs. The maximal rate of transcription occurred late in the growth cycle from a single but highly conserved promoter which had close similarity with the Escherichia coli consensus promoter sequences. A stem and loop structure which could be involved in regulating translation of mRNA occurred in all three species between the transcriptional start point and the ribosome binding site. Although transcription is initiated from a single promoter in all three chlamydial species, transcriptional termination points for the monocistronic and bicistronic mRNAs differ in both number and position.

Summary: pJV1 is an 11 kb, high-copy-number conjugative Streptomyces phaeochromogenes plasmid that replicates by the rolling circle mechanism (RCR). Sequencing combined with functional analysis of deletion, insertion and frameshift mutations was used to characterize the genes involved in plasmid transfer and chromosome mobilization (Cma), the single-strand origin for RCR and an associated strong incompatibility (Sti) determinant. pJV1 contains two essential transfer genes whose expression is regulated by an adjacent repressor gene with similarity to the GntR family of regulators. A consensus sequence specific for the helix-turn-helix motifs of repressor proteins of Streptomyces plasmids is proposed. Unregulated expression of the transfer genes by inactivation of the repressor is lethal. Three additional genes increase intramycelial plasmid spread resulting in pock formation but, unlike the essential transfer genes, are not required for Cma. The pJV1 transfer genes and their regulatory region, but not the minimal replication region encoding the double-strand replication origin and replication protein, are similar in their sequence and arrangement to those of the Streptomyces nigrifaciens plasmid pSN22, revealing a modular organization of Streptomyces RCR plasmids.

Summary: Streptomyces griseus ATCC 12475 fails to produce streptomycin when grown at 34 °C or above, although growth is appreciable up to at least 37 °C. This depression of streptomycin production at elevated growth temperature is manifest equally in liquid and on solid, and with complex and minimal, media. We report studies with gene fusions of the reporter genes aph or xylE to restriction fragments containing the streptomycin biosynthesis promoter PstrB1. aph constructs were in high, and xylE constructs in low, copy number vectors. Two strB1 promoter fragments were used, one requiring activation by the pathway-specific activator StrR of S. griseus, the other reportedly activator independent. PstrB1 expression in the aph constructs in S. griseus and in S. lividans was significantly reduced at 37 °C compared to 30 °C. Some of this reduction could be explained by lower plasmid copy number at the higher temperature, but strR-dependent expression was clearly temperature controlled. Using the xylE reporter system, the temperature dependence of PstrB1 expression was confirmed but, surprisingly, the strR dependence of the two promoter fragments differed from that observed in the multicopy aph constructs. These data identify a temperature-dependent promoter which may contribute to the depressive effect of elevated growth temperature on streptomycin production.

Summary: In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5′-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in αrdB, the principal . factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNALeu for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.

Summary: Bordetella pertussis produces a number of virulence factors whose expression is coordinately regulated by the bvgAS locus. Transcription of virulence genes is repressed by environmental factors such as low temperature (25°C) and chemical stimuli. Temperature shift of bacterial cultures from 25°C to 37°C activates two classes of bvg-regulated virulence genes: the early genes, which are activated within 10 min, and late genes, which require 2-4 h for activation. During the interval between the activation of the early and late genes, the intracellular concentration of BvgA increases 50-fold. It has been proposed that this increased concentration may be required for the activation of the late genes. Here we have analysed the response of the bvg locus to intermediate temperatures and to repeated temperature shifts. Temperature shifts of B. pertussis cultures from 22°C to 28°C, 32°C or 35°C resulted in the synthesis of low, intermediate, and high amounts of BvgA. This implied that the intracellular concentration of BvgA is temperature-dependent. We have also observed that the amount of virulence factors produced correlates with the BvgA concentration. When bacteria grown at 37°C were shifted to 22 °C, transcription from the adenylate cyclase toxin haemolysis promoter (PAC) was repressed after 30 min, while transcription from the bvg (P1) and filamentous haemagglutinin (PFHA) promoters was repressed after 2 h. During this time, the amount of BvgA did not decrease. A subsequent temperature shift from 22°C to 37°C induced transcription from the P, and PFHA promoters after 10 min and transcription from the PAC promoter after 20 min. This result shows that in the presence of a high concentration of BvgA, the time lag between temperature shift and late promoter transcription is reduced from 2-4 h to 20 min. The above data support the proposal that the concentration of BvgA plays a role in activating expression of the late genes.

Summary: A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli and clones were screened for a bacteriocin 28b insensitive phenotype. One clone was found that showed partial resistance to bacteriocin 28b. By using Tn5tac1 insertions it was shown that this phenotype was due to the expression in E. coli of an outer-membrane protein of 17 kDa (Omp4). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 515 bp. The deduced amino acid sequence has 172 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues and, when compared to other enterobacterial outer-membrane proteins, most closely resembles a family of small outer-membrane proteins of Enterobacteriaceae whose known functions appear to be related with virulence. Immunoblotting experiments showed that Omp4 is present in 15 biotypes of S. marcescens. The bacteriocin 28b resistance phenotype conferred on E. coli by Omp4 appears to be pleiotropic since overexpression of the Omp4-encoding gene leads to a decrease in the amount of OmpA, OmpF and/or OmpC; OmpA and OmpF are the receptors for bacteriocin 28b in E. coli.

Summary: Nucleotide sequence analysis of the mxcQ and mxcE loci, required for the synthesis of methanol dehydrogenase in Methylobacterium organophilum XX, has revealed two open reading frames that show significant similarity to sequences of prokaryotic two-component systems, especially MxaY and MxaX proteins of another methylotrophic bacterium, Paracoccus denitrificans. Cell-free extracts and DNA-column-fractionated proteins from wild-type M. organophilum XX cells grown on methanol or succinate contained protein(s) that were able to bind specifically to the upstream region of methanol dehydrogenase large subunit gene (mxaF). In contrast, cell-free extracts from mxcQ and mxcE mutant strains of M. organophilum XX had zero or reduced binding activity towards the promoter fragments of the mxaF gene. This is consistent with the involvement of the mxcQ and mxcE genes in transcriptional regulation of methanol dehydrogenase synthesis. Analyses of sequential deletions of the mxaF upstream region have defined the functional boundary of the promoter/operator region of this gene and identified one nucleotide segment as essential to the activation of mxaF.

Summary: Genes encoding -ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.

Summary: A complete isoleucyl-tRNA synthetase gene (ileS) of Campylobacter jejuni was isolated from a C. jejuni TGH9011 genomic DNA library constructed in pBluescript. The complete coding sequence, flanking regions and transcription start point were determined. The deduced isoleucyl-tRNA synthetase (lleRS) had 917 amino acids with a molecular mass of 105399 Da, which was consistent with the observed size of 105 kDa in Escherichia coli maxicells. The ileS gene was mapped onto the physical map of the C. jejuni genome. Alignment of the C. jejuni lleRS sequence with six other bacterial lleRS sequences and two lower eukaryotic lleRS sequences identified seven conserved motifs, including the two signature sequences, HIGH and KMSKS, of class I aminoacyl-tRNA synthetases.

Summary: The structural gene prtVp encoding the extracellular protease of Vibrio parahaemolyticus strain 93 was cloned in Escherichia coli and sequenced. The cloned DNA fragment contained a 1761 bp ORF encoding a 587 amino acid protein. The deduced polypeptide is composed of a 25 amino acid signal peptide and a 562 amino acid extracellular polypeptide with a calculated molecular mass of 63 156 Da. Protease analysis using a gelatin-containing SDS-polyacrylamide gel detected the presence of a 62 kDa protease that was present in the culture supernatant fractions of the wild-type V. parahaemolyticus strain and of E. coli bearing a pUC119 recombinant with the prtVp DNA insert. The protease activity was inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, which suggested that it is a metalloprotease. The deduced amino acid sequence of PrtVp has 32% identity with that of the collagenase of Vibrio alginolyticus, but has no identity with those of the bacterial proteases. A conserved zinc-binding domain was also found in PrtVp from homology comparison with other metalloproteases. This PrtVp can cause weak haemolysis on blood agar.

Summary: Monocins in Listeria were induced by UV-irradiation of liquid cultures, and defective phage particles were purified from the lysates. Electron microscopy showed flexible, non-contractile bacteriophage-tail-like particles, consisting of specific proteins of molecular mass 20-45 kDa and pl 4.6-6.7. These particles were able to lyse listerial cells. DNA sequence homologies between chromosomal DNA of monocin-producing strains and labelled Listeria phage DNAs were inferred from DNA/DNA hybridizations, suggesting that most of the prophage DNA is still present in the listerial chromosome. An endolysin gene cpl2438 was cloned from listerial chromosomal DNA and was identified by its expression of lytic activity against Listeria cells in a bioassay. The gene consists of 864 nt encoding a protein of 287 aa with a calculated molecular mass of 32975 Da (CPL2438). This is in good agreement with the size of a protein observed in SDS-PAGE after overexpression of the lytic protein in Escherichia coli. The nucleotide sequence of a putative holin gene (hol2438, 291 nt) upstream of cpl2438 was determined after PCR-amplification of listerial DNA and it shows typical features common to the holin gene family. Expression of the encoded protein (HOL2438, 95 aa, 10.1 kDa) in E. coli was found to be lethal for the host cells. The results underline the close relationship between monocins and intact Listeria bacteriophages, indicating that monocins are incompletely assembled phage particles derived from cryptic prophages of Listeria, probably including the phage lysin.