Unified Workflow for the Rapid and In-Depth Characterization of Bacterial Proteomes.

Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data analysis strategies in bacterial proteomics. We investigated workflow performances on six representative species with highly different physiologic properties to mimic bacterial diversity. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in-solution digest. Peptides were separated on a 30-min linear microflow liquid chromatography gradient and analyzed in data-independent acquisition mode. Data analysis was performed with DIA-NN using a predicted spectral library. Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs, and biological safety. With this rapid workflow, over 40% of all encoded genes were detected per bacterial species. We demonstrated the general applicability of our workflow on a set of 23 taxonomically and physiologically diverse bacterial species. We could confidently identify over 45,000 proteins in the combined dataset, of which 30,000 have not been experimentally validated before. Our work thereby provides a valuable resource for the microbial scientific community. Finally, we grew Escherichia coli and Bacillus cereus in replicates under 12 different cultivation conditions to demonstrate the high-throughput suitability of the workflow. The proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom.

Pseudomonas cremoris sp. nov., a novel proteolytic species isolated from cream.

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize the novel strains and to investigate their taxonomic status. Examinations based on the topology of core genome phylogenomy as well as average nucleotide identity (ANIm) comparisons suggested a novel Pseudomonas species within the Pseudomonas fluorescens subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106T was most closely related to Pseudomonas nabeulensis CECT 9765T and Pseudomonas kairouanensis CECT 9766T. The G+C content of strain WS 5106T was 60.1 mol%. Morphologic analyses revealed Gram-stain-negative, aerobic, catalase and oxidase positive, rod-shaped and motile cells. Proteolysis on skimmed milk agar as well as lipolysis on tributyrin agar occurred at both 28 and 6 °C. Tolerated growth conditions were temperatures between 4 and 34 °C, pH values between 6.0 and 8.0, and salt concentrations of up to 5 %. Fatty acid profiles showed a pattern typical for Pseudomonas, with C16 : 0 as the dominant component. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the dominating quinone was Q-9. Based on these results, it is proposed to classify the strains as a novel species, Pseudomonas cremoris sp. nov., with WS 5106T (=DSM 111143T=LMG 31863T) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.

Pseudomonas saxonica sp. nov., isolated from raw milk and skimmed milk concentrate.

A polyphasic approach was used to investigate the taxonomic status of two bacterial strains, WS 5072T and WS 5092, isolated from skimmed milk concentrate and raw cow's milk. The 16S rRNA and rpoD gene sequences affiliated the strains to the same, hitherto unknown, Pseudomonas species. Further examinations of the draft genomes based on multilocus sequence analysis and average nucleotide identity confirmed the presence of a novel Pseudomonas species. It was most closely related to Pseudomonas fragi DSM 3456T with 86.3 % ANIm. The DNA G+C content of strain WS 5072T was 56.3 mol%. Cells were aerobic, Gram-negative, catalase and oxidase positive, rod-shaped and motile. Growth occurred at 4-34 °C, pH 5.5-8.0 and with salt concentrations of up to 7 %. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The dominating quinone was Q-9 with 94 %, with noticeable amounts of Q-8 (5 %) and traces of Q-7 and Q-10. Fatty acid profiles showed a composition common for Pseudomonas with the major component C16 : 0. Based on these results, the novel species Pseudomonas saxonica sp. nov. is proposed, with the type strain WS 5072T (=DSM 108989T=LMG 31234T) and the additional strain WS 5092 (=DSM 108990=LMG 31235).

Reprogramming Human Siderocalin To Neutralize Petrobactin, the Essential Iron Scavenger of Anthrax Bacillus.

Bacillus anthracis owes its pronounced virulence-apart from specific toxins-to a twofold import mechanism for FeIII ions. This pathogenic bacterium secretes the siderophores bacillibactin (BB) and petrobactin (PB), of which only BB is neutralized by human siderocalin, an abundant lipocalin in plasma. We describe its reshaping via combinatorial protein design to bind PB⋅FeIII instead of BB⋅FeIII , and with even higher affinity (KD ≈20 pm). X-ray crystallographic analysis of the resulting "petrocalin" in complex with PB⋅GaIII reveals a positively charged ligand pocket while the extended butterfly-like conformation of the bound PB provides a rationale for the missing recognition by the natural siderocalin. In microbiological studies, a combination of petrocalin and siderocalin effectively suppressed the growth of a BB+ /PB+ strain of Bacillus cereus under iron-limiting culture conditions. Thus, our reprogrammed lipocalin may offer novel treatment options for devastating infections caused by B. anthracis.

Differentiation of ncRNAs from small mRNAs in Escherichia coli O157:H7 EDL933 (EHEC) by combined RNAseq and RIBOseq - ryhB encodes the regulatory RNA RyhB and a peptide, RyhP.

BACKGROUND: While NGS allows rapid global detection of transcripts, it remains difficult to distinguish ncRNAs from short mRNAs. To detect potentially translated RNAs, we developed an improved protocol for bacterial ribosomal footprinting (RIBOseq). This allowed distinguishing ncRNA from mRNA in EHEC. A high ratio of ribosomal footprints per transcript (ribosomal coverage value, RCV) is expected to indicate a translated RNA, while a low RCV should point to a non-translated RNA. RESULTS: Based on their low RCV, 150 novel non-translated EHEC transcripts were identified as putative ncRNAs, representing both antisense and intergenic transcripts, 74 of which had expressed homologs in E. coli MG1655. Bioinformatics analysis predicted statistically significant target regulons for 15 of the intergenic transcripts; experimental analysis revealed 4-fold or higher differential expression of 46 novel ncRNA in different growth media. Out of 329 annotated EHEC ncRNAs, 52 showed an RCV similar to protein-coding genes, of those, 16 had RIBOseq patterns matching annotated genes in other enterobacteriaceae, and 11 seem to possess a Shine-Dalgarno sequence, suggesting that such ncRNAs may encode small proteins instead of being solely non-coding. To support that the RIBOseq signals are reflecting translation, we tested the ribosomal-footprint covered ORF of ryhB and found a phenotype for the encoded peptide in iron-limiting condition. CONCLUSION: Determination of the RCV is a useful approach for a rapid first-step differentiation between bacterial ncRNAs and small mRNAs. Further, many known ncRNAs may encode proteins as well.

Chemodiversity of cereulide, the emetic toxin of Bacillus cereus.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans.

BACKGROUND: The saprophytic pathogen Listeria monocytogenes has to cope with a variety of acidic habitats during its life cycle. The impact of low-temperature coupled with pH decrease for global gene expression and subsequent virulence properties, however, has not been elucidated. RESULTS: qRT-PCR revealed for the first time a transient, acid triggered prfA induction of approximately 4-fold, 5.7-fold, 7-fold and 9.3-fold 60 to 90 min after acid shock of L. monocytogenes at 37°C, 25°C, 18°C, and 10°C, respectively. Comparable data were obtained for seven different L. monocytogenes strains, demonstrating that prfA induction under these conditions is a general response of L. monocytogenes. Transcriptome analysis revealed that the in vivo-relevant genes bsh, clpP, glpD, hfq, inlA, inlB, inlE, lisR, and lplA1 as well as many other genes with a putative role during infection are transiently induced upon acid shock conducted at 25°C and 37°C. Twenty-five genes repressed upon acid shock are known to be down regulated during intracellular growth or by virulence regulators. These data were confirmed by qRT-PCR of twelve differentially regulated genes and by the identification of acid shock-induced genes influenced by σB. To test if up regulation of virulence genes at temperatures below 37°C correlates with pathogenicity, the capacity of L. monocytogenes to invade epithelial cells after acid shock at 25°C was measured. A 12-fold increased number of intracellular bacteria was observed (acid shock, t = 60 min) that was reduced after adaptation to the level of the unshocked control. This increased invasiveness was shown to be in line with the induction of inlAB. Using a nematode infection assay, we demonstrated that Caenorhabditis elegans fed with acid-shocked L. monocytogenes exhibits a shorter time to death of 50% (TD50) of the worms (6.4 days) compared to infection with unshocked bacteria (TD50 = 10.2 days). CONCLUSIONS: PrfA and other listerial virulence genes are induced by an inorganic acid in a temperature-dependent manner. The data presented here suggest that low pH serves as a trigger for listerial pathogenicity at environmental temperatures.

Micrococcus cohnii sp. nov., isolated from the air in a medical practice.

Three Gram-reaction-positive bacteria, isolated from the air in a medical practice (strains WS4601(T), WS4602) or a pharmaceutical clean room (strain WS4599), were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences of the three novel strains showed that they formed a distinct lineage within the genus Micrococcus, sharing 16S rRNA gene sequence similarities of 96.1-98.0 % with other species of this genus. Chemotaxonomic features also supported the classification of the three novel strains within the genus Micrococcus. The major cellular fatty acids of strain WS4601(T) were anteiso-C(15 : 0) and iso-C(15 : 0), the cell-wall peptidoglycan was of type A3α (L-Lys-L-Ala), and the predominant respiratory quinones were MK-7(H(2)) and MK-8(H(2)). The polar lipid profile contained diphosphatidylglycerol and phosphatidylglycerol, but no phosphatidylinositol. The G+C content of the genomic DNA was 70.4 mol%. Numerous physiological properties were found that clearly distinguished strains WS4599, WS4601(T) and WS4602 from established members of the genus Micrococcus. Based on the phenotypic and phylogenetic data, strains WS4599, WS4601(T) and WS4602 are considered to represent three different strains of a novel species of the genus Micrococcus, for which the name Micrococcus cohnii sp. nov. is proposed. The type strain is WS4601(T) (=DSM 23974(T)=LMG 26183(T)).

Domibacillus robiginosus gen. nov., sp. nov., isolated from a pharmaceutical clean room.

A novel red-pigmented bacterial strain, designated WS 4628(T), was isolated from a pharmaceutical clean room of a vaccine-producing company and was investigated in a taxonomic study using a polyphasic approach. The strain was Gram-stain-positive, strictly aerobic, motile, catalase-positive and produced spherical to slightly ellipsoidal endospores in rods. The genomic DNA G+C content was 44.1 mol%. The major fatty acids were anteiso-C15:0, iso-C15:0 and anteiso-C17:0 and the predominant quinone was MK-6. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, an unidentified phosphoglycolipid and an unidentified phospholipid. meso-diaminopimelic acid (type A1γ) was present in the cell-wall peptidoglycan and the major whole-cell sugars were glucose and ribose. The closest phylogenetic neighbours were identified as Bacillus badius ATCC 14574(T) (95.8% 16S rRNA gene sequence similarity), Bacillus indicus Sd/3(T) (94.8%), Jeotgalibacillus alimentarius YKJ-13(T) (94.8%) and Bacillus cibi JG-30(T) (94.8%). Phylogenetic, physiological, biochemical and morphological differences between strain WS 4628(T) and its closest relatives in the families Bacillaceae and Planococcaceae suggest that this strain represents a novel species in a new genus in the family Bacillaceae for which the name Domibacillus robiginosus gen. nov., sp. nov. is proposed; the type strain of the type species is WS 4628(T) (=DSM 25058(T)=LMG 26645(T)).

Naumannella halotolerans gen. nov., sp. nov., a Gram-positive coccus of the family Propionibacteriaceae isolated from a pharmaceutical clean room and from food.

Four Gram-stain-positive, aerobic bacterial strains isolated from a pharmaceutical clean room (strain WS4616(T)), a dessert milk product (strain WS4617) and from raw milk (strains WS4623 and WS4624) were characterized using a polyphasic approach. Phylogenetic analyses based on 16S rRNA and recA gene sequences showed that they formed a distinct lineage within the family Propionibacteriaceae. Similarity values between 16S rRNA gene sequences of the four novel strains and the type species of all genera belonging to the family Propionibacteriaceae were 89.2-94.1%. The major cellular fatty acid was anteiso-C(15:0) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Respiratory quinones were MK-8(H(4)) and MK-9(H(4)). The cell-wall peptidoglycan of type A3γ contained ll-diaminopimelic acid, alanine, glycine and glutamic acid. The G+C content of the genomic DNA of strain WS4616(T) was 67.7 mol%. The whole-cell sugar pattern contained ribose, mannose, arabinose, glucose and galactose. On the basis of phenotypic and genetic data, strains WS4616(T), WS4617, WS4623 and WS4624 are classified as members of a novel species in a new genus of the family Propionibacteriaceae, for which the name Naumannella halotolerans gen. nov., sp. nov. is proposed. The type strain is WS4616(T) ( = DSM 24323(T) = LMG 26184(T)) and three additional strains are WS4617, WS4623 and WS4624.

Inhibition of cereulide toxin synthesis by emetic Bacillus cereus via long-chain polyphosphates.

Severe intoxications caused by the Bacillus cereus emetic toxin cereulide can hardly be prevented due to the ubiquitous distribution and heat resistance of spores and the extreme thermal and chemical stability of cereulide. It would therefore be desirable to inhibit cereulide synthesis during food manufacturing processes or in prepared foods, which are stored under time-temperature abuse conditions. Toward this end, the impacts of three long-chain polyphosphate (polyP) formulations on growth and cereulide production were examined. The inhibition was dependent on the concentration and the type of the polyP blend, indicating that polyPs and not the orthophosphates were effective. Quantitative PCR (qPCR) monitoring at sublethal concentrations revealed that polyPs reduced the transcription of ces nonribosomal peptide synthetase (NRPS) genes by 3- to 4-fold along with a significantly reduced toxin production level. At lower concentrations, toxin synthesis was decreased, although the growth rate was not affected. These data indicate a differential effect on toxin synthesis independent of growth inhibition. The inhibition of toxin synthesis in food was also observed. Despite the growth of B. cereus, toxin synthesis was reduced by 70 to 100% in two model food systems (reconstituted infant food and oat milk), which were analyzed with HEp-2 cell culture assays and high-performance liquid chromatography (HPLC)/electrospray ionization-time of flight mass spectrometry (ESI-TOF-MS). Accordingly, ces promoter activity was strongly downregulated, as visualized by using a lux-based reporter strain. These data illustrate the potential of polyphosphate formulations to reduce the risk of cereulide synthesis in food and may contribute to targeted hurdle concepts.

Both thiamine uptake and biosynthesis of thiamine precursors are required for intracellular replication of Listeria monocytogenes.

Thiamine pyrophosphate is an essential cofactor involved in central metabolism and amino acid biosynthesis and is derived from thiamine (vitamin B(1)). The extent to which this metabolite is available to bacterial pathogens replicating within host cells is still little understood. Growth studies using modified minimal Welshimer's broth (mMWB) supplemented with thiamine or the thiamine precursor hydroxymethylpyrimidine (HMP) showed that Listeria monocytogenes, in agreement with bioinformatic prediction, is able to synthesize thiamine only in the presence of HMP. This appears to be due to a lack of ThiC, which is involved in HMP synthesis. The knockout of thiD (lmo0317), which probably catalyzes the phosphorylation of HMP, inhibited growth in mMWB supplemented with HMP and reduced the replication rate of L. monocytogenes in epithelial cells. Mutation of a predicted thiamine transporter gene, lmo1429, led to reduced proliferation of L. monocytogenes in mMWB containing thiamine or thiamine phosphates and also within epithelial cells but had no influence on the expression of the virulence factors Hly and ActA. The toxic thiamine analogue pyrithiamine inhibited growth of wild-type strain EGD but not of the transporter mutant EGDDeltathiT. We also demonstrated that ThiT binds thiamine, a finding compatible with ThiT acting as the substrate-binding component of a multimeric thiamine transporter complex. These data provide experimental evidence that Lmo1429 homologs including Bacillus YuaJ are necessary for thiamine transport in gram-positive bacteria and are therefore proposed to be annotated "ThiT." Taken together, these data indicate that concurrent thiamine uptake and biosynthesis of thiamine precursors is a strategy of L. monocytogenes and possibly other facultative intracellular pathogens to enable proliferation within the cytoplasm.

Insecticidal genes of Yersinia spp.: taxonomical distribution, contribution to toxicity towards Manduca sexta and Galleria mellonella, and evolution.

BACKGROUND: Toxin complex (Tc) proteins termed TcaABC, TcdAB, and TccABC with insecticidal activity are present in a variety of bacteria including the yersiniae. RESULTS: The tc gene sequences of thirteen Yersinia strains were compared, revealing a high degree of gene order conservation, but also remarkable differences with respect to pseudogenes, sequence variability and gene duplications. Outside the tc pathogenicity island (tc-PAIYe) of Y. enterocolitica strain W22703, a pseudogene (tccC2'/3') encoding proteins with homology to TccC and similarity to tyrosine phosphatases at its C-terminus was identified. PCR analysis revealed the presence of the tc-PAIYe and of tccC2'/3'-homologues in all biotype 2-5 strains tested, and their absence in most representatives of biotypes 1A and 1B. Phylogenetic analysis of 39 TccC sequences indicates the presence of the tc-PAIYe in an ancestor of Yersinia. Oral uptake experiments with Manduca sexta revealed a higher larvae lethality of Yersinia strains harbouring the tc-PAIYe in comparison to strains lacking this island. Following subcutaneous infection of Galleria mellonella larvae with five non-human pathogenic Yersinia spp. and four Y. enterocolitica strains, we observed a remarkable variability of their insecticidal activity ranging from 20% (Y. kristensenii) to 90% (Y. enterocolitica strain 2594) dead larvae after five days. Strain W22703 and its tcaA deletion mutant did not exhibit a significantly different toxicity towards G. mellonella. These data confirm a role of TcaA upon oral uptake only, and suggest the presence of further insecticidal determinants in Yersinia strains formerly unknown to kill insects. CONCLUSION: This study investigated the tc gene distribution among yersiniae and the phylogenetic relationship between TccC proteins, thus contributing novel aspects to the current discussion about the evolution of insecticidal toxins in the genus Yersinia. The toxic potential of several Yersinia spp. towards M. sexta and G. mellonella demonstrated here for the first time points to insects as a natural reservoir for yersiniae.

Low temperature-induced insecticidal activity of Yersinia enterocolitica.

The insecticidal toxin complexes (Tcs) are produced by several Enterobacteriaceae associated with insects, such as Photorhabdus luminescens, Serratia entomophila and Xenorhabdus nematophilus. Genome sequences revealed tc-like genes in Yersinia spp., but insecticidal activity of this genus associated with the toxins has not been described. Through the search for genes upregulated at low growth temperatures in Yersinia enterocolitica strain W22703, a genomic island of 19 kb termed tc-PAI(Ye) with homologues of the toxin genes tcaA, tcaB, tcaC and tccC was identified. Southern blot and polymerase chain reaction (PCR) analysis of 34 strains demonstrated that the tc-PAI(Ye) is present in biovars 2, 3 and 4, but neither in biovars 1A and 1B, nor in five Yersinia species apathogenic in humans. Using the luxCDABE operon as reporter, the expression of the toxin genes was shown to be completely repressed in cells cultured at 37 degrees C, and to increase by 4.6 orders of magnitude when the growth temperature was decreased gradually to 10 degrees C. These data provide the first indication that temperature is a critical parameter for induction or repression of tc gene transcription. Whole-cell extracts of Y. enterocolitica strain W22703 cultivated at 10 degrees C, but not at 30 degrees C, led to insect mortality when fed to Manduca sexta larvae, in contrast to an insertional tcaA mutant. Overall the results suggest that the tc-PAI(Ye) could play an important role in the transmission and survival of pathogenic Y. enterocolitica strains outside mammalian hosts.

UV irradiation and desiccation modulate the three-dimensional extracellular matrix of Nostoc commune (Cyanobacteria).

Cyanobacterium Nostoc commune can tolerate the simultaneous stresses of desiccation, UV irradiation, and oxidation. Acidic WspA, of approximately 33.6 kDa, is secreted to the three-dimensional extracellular matrix and accounts for greater than 70% of the total soluble protein. The wspA gene of N. commune strain DRH1 was cloned and found in a single genomic copy, in a monocistronic operon. Transcription of wspA and sodF (superoxide dismutase), and synthesis and secretion of WspA, were induced upon desiccation or UV-A/B irradiation of cells. Recombinant WspA binds the UV-A/B absorbing pigment scytonemin through non-covalent interactions. WspA peptide polymorphism, and heterogeneity of multiple wspA sequences within cells of a single colony, account for distinct WspA isoforms. WspA has no similarity to entries in the sequence databases and wspA, a possible xenolog, is restricted to a subset of strains in the "form species" N. commune characterized through group I intron phylogeny. We hypothesize that WspA plays a central role in the global stress response of N. commune through modulation of the structure and function of the three-dimensional extracellular matrix, particularly the transport, distribution, and/or macromolecular architecture of mycosporine and scytonemin UV-A/B absorbing pigment complexes.

Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains.

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.

Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain.

We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118. Native hol118 was cloned into lambdaDeltaSthf, devoid of the S holin, and tested in an E. coli background. Surprisingly, it caused very late cell lysis, beginning at 80 min after induction. Immunological analyses demonstrated that Hol118 appears in the cytoplasmic membrane shortly after infection. The hol118 gene features a dual start motif similar to lambda S. Therefore, different N-terminally modified Hol118 variants were tested. However, in contrast to lambda S, inactivation of AUG-1 or AUG-2 showed no significant influence on lysis timing. In addition, Hol118-mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S. Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG-3) at nucleotide position 40. We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full-length polypeptide. However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support lambda R-mediated lysis. In contrast, specific mutations introduced to abolish translation initiation at AUG-3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83). This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans. A deviation from the lambda S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118. Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by lambda.

Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation.

Two temperate viruses, phi3626 and phi8533, have been isolated from lysogenic Clostridium perfringens strains. Phage phi3626 was chosen for detailed analysis and was inspected by electron microscopy, protein profiling, and host range determination. For the first time, the nucleotide sequence of a bacteriophage infecting Clostridium species was determined. The virus belongs to the Siphoviridae family of the tailed phages, the order Caudovirales. Its genome consists of a linear double-stranded DNA molecule of 33,507 nucleotides, with invariable 3'-protruding cohesive ends of nine residues. Fifty open reading frames were identified, which are organized in three major life cycle-specific gene clusters. The genes required for lytic development show an opposite orientation and arrangement compared to the lysogeny control region. A function could be assigned to 19 gene products, based upon bioinformatic analyses, N-terminal amino acid sequencing, or experimental evidence. These include DNA-packaging proteins, structural components, a dual lysis system, a putative lysogeny switch, and proteins that are involved in replication, recombination, and modification of phage DNA. The presence of genes encoding a putative sigma factor related to sporulation-dependent sigma factors and a putative sporulation-dependent transcription regulator suggests a possible interaction of phi3626 with onset of sporulation in C. perfringens. We found that the phi3626 attachment site attP lies in a noncoding region immediately downstream of int. Integration of the viral genome occurs into the bacterial attachment site attB, which is located within the 3' end of a guaA homologue. This essential housekeeping gene is functionally independent of the integration status, due to reconstitution of its terminal codons by phage sequence.

Intraspecific diversity of Brevibacterium linens, Corynebacterium glutamicum and Rhodococcus erythropolis based on partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy.

The intraspecific diversity of 31 strains of Brevibacterium linens, 27 strains of Corynebacterium glutamicum and 29 strains of Rhodococcus erythropolis was determined by partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. As a prerequisite for the analyses, 27 strains derived from culture collections which had carried invalid or wrong species designations were reclassified in accordance with polyphasic taxonomical data. FT-IR spectroscopy proved to be a rapid and reliable method for screening for similar isolates and for identifying these actinomycetes at the species level. Two main conclusions emerged from the analyses. (1) Comparison of intraspecific 16S rDNA similarities suggested that R. erythropolis strains have a very low diversity, B. linens displays high diversity and C. glutamicum occupies an intermediate position. (2) No correlation of FT-IR spectral similarity and 16S rDNA sequence similarity below the species level (i.e. between strains of one species) was observed. Therefore, diversification of 16S rDNA sequences and microevolutionary change of the cellular components detected by FT-IR spectroscopy appear to be de-coupled.

Long-Chain Polyphosphates Inhibit Growth of Clostridium tyrobutyricum in Processed Cheese Spreads.

The effect of novel food-grade long-chain polyphosphate formulations (JOHA HBS sodium polyphosphate glassy, 69 ± 1% P2O5, and two similar salts (HBS-1 and HBS-9) on the growth of Clostridium tyrobutyricum ATCC 25755 in liquid culture and in pasteurized, processed cheese spreads was evaluated. In broth, 0.1 % polyphosphate was sufficient to inhibit vegetative growth of the organism. In addition, a panel of 21 other gram-positive and 11 gram-negative bacteria were tested for their sensitivity against the polyphosphates. Whereas 17 of the gram-positives could be inhibited by 0.05 to 0.3% polyphosphate, none of the tested gram-negatives were affected. Two different cheese spread formulations (cheese blend A: 55% moisture, 47.2% fat in dry matter; cheese blend B: 55% moisture, 57% fat in dry matter) were fortified with 0.1 % to 1.0% polyphosphates, inoculated with 5 × 105 (cheese blend A) or 2.5 × 106 (cheese blend B) C. tyrobutyricum spores per gram, and incubated at 35°C for up to 7 weeks. Determination of viable cell counts was carried out at days 1, 9, 19, and 49 (cheese blend A) and 8, 16, 27, 35, and 50 (cheese blend B). While 0.1 % polyphosphate had little effect, higher concentrations were increasingly inhibitory to growth from a spore inoculum, to cell multiplication, and to gas formation. With 0.5% polyphosphate, onset of growth was delayed for about 3 weeks in cheese blend A, while this concentration was able to inhibit the organism in cheese blend B. In view of the experimental parameters selected (high initial contamination level; intrinsic and extrinsic parameters optimized for growth of clostridia), 0.5% polyphosphate may be sufficient to control C. tyrobutyricum growth under "normal" conditions, where initial spore counts are rather low, and storage temperatures are usually at or below 20°C. Moreover, clostridia were completely inhibited by 1.0% polyphosphate, which clearly indicated the usefulness of these polyphosphates for prevention of butyric blowing in pasteurized processed cheese spreads.