- Volume 158, Issue 6, 2012

The mechanisms that allow Streptococcus pyogenes to survive and persist in the human host, often in spite of antibiotic therapy, remain poorly characterized. Therefore, the determination of culture conditions for long-term studies is crucial to advancement in this field. Stationary cultures of S. pyogenes strain NZ131 and its spontaneous small-colony variant OK171 were found to survive in rich medium for less than 2 weeks, and this inability to survive resulted from the acidification of the medium to below pH 5.5, which the cells did not tolerate for longer than 6–7 days. The growth of NZ131 resulted in acidification of the culture to below pH 5.5 by the onset of stationary phase, and the loss of viability occurred in a linear fashion. These results were also found to be true for M49 strain CS101 and for M1 strain SF370. The S. pyogenes strains could be protected from killing by the addition of a buffer that stabilized the pH of the medium at pH 6.5, ensuring bacterial survival to at least 70 days. By contrast, increasing the glucose added to the medium accelerated the loss of culture viability in strain NZ131 but not OK171, suggesting that the small-colony variant is altered in glucose uptake or metabolism. Similarly, acidification of the medium prior to inoculation or at the middle of exponential phase resulted in growth inhibition of all strains. These results suggest that control of the pH is crucial for establishing long-term cultures of S. pyogenes.

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium in which certain vegetative cells differentiate into heterocysts, which are specialized cells for nitrogen fixation. Heterocysts are unable to carry out photosynthesis and are supplied with carbohydrate required for nitrogen fixation from neighbouring vegetative cells. Thus, filament integrity is very important for diazotrophic growth of the heterocystous cyanobacteria. The pknH gene (alr1336), encoding a putative Ser/Thr protein kinase, was upregulated in heterocysts after nitrogen deprivation. Its expression was developmentally regulated by the hetR gene. Expression levels of genes involved in heterocyst maturation, such as hepA, hglE and nifH, in the pknH disruptant were similar to those of the wild-type strain. The disruptant was able to form heterocysts with nitrogenase activity, but most heterocysts were detached from filaments. Hence, the pknH disruptant showed a growth defect in the medium without combined nitrogen. It is concluded that the pknH gene is not involved in the development of heterocyst function but is involved in maintaining connections between heterocysts and vegetative cells.

Flagella provide advantages to Escherichia coli by facilitating taxis towards nutrients and away from unfavourable niches. On the other hand, flagellation is an energy sink to the bacterial cell, and flagella also stimulate host innate inflammatory responses against infecting bacteria. The flagellar assembly pathway is ordered and under a complex regulatory circuit that involves three classes of temporally regulated promoters as well as the flagellar master regulator FlhD4C2. We report here that transcription of the flhDC operon from the class 1 promoter is under negative regulation by MatA, a key activator of the common mat (or ecp) fimbria operon that enhances biofilm formation by E. coli. Ectopic expression of MatA completely precluded motility and flagellar synthesis in the meningitis-associated E. coli isolate IHE 3034. Northern blotting, analysis of chromosomal promoter–lacZ fusions and electrophoretic mobility shift assays revealed an interaction between MatA and the flhDC promoter region that apparently repressed flagellum biosynthesis. However, inactivation of matA in the chromosome of IHE 3034 had only a minor effect on flagellation, which underlines the complexity of regulatory signals that promote flagellation in E. coli. We propose that the opposite regulatory actions of MatA on mat and on flhDC promoters advance the adaptation of E. coli from a planktonic to an adhesive lifestyle.

Replication of staphylococcal multiresistance plasmid pSK41 is initiated by binding of the replication initiator protein (Rep) to the Rep boxes, a series of four direct repeats located centrally within the rep gene. A Staphylococcus aureus strain was engineered to provide Rep in trans, allowing localization of the pSK41 origin of replication (oriV) to a 185 bp segment, which included the Rep boxes and a series of downstream direct repeats. Deletion analysis of individual Rep boxes revealed that all four Rep boxes are required for maximum origin activity, with the deletion of one or more Rep boxes having a significant effect on the proficiency of replication. However, a hierarchy of importance was identified among the Rep boxes, which appears to be mediated by the minor sequence variations that exist between them. DNA binding studies with truncated Rep proteins have enabled the DNA binding domain to be localized to the N-terminal 134 amino acids of the protein.

The conventional model of Rho-dependent transcription termination in bacteria requires RNA-dependent translocase activity of the termination factor Rho as well as many kinetically controlled steps to execute efficient RNA release from the transcription elongation complex (EC). The involvement of the kinetically controlled steps, such as RNA binding, translocation and RNA release from the EC, means that this termination process must be kinetically coupled to the transcription elongation process. The existence of these steps in vivo has not previously been delineated in detail. Moreover, the requirement for translocase activity in Rho-dependent termination has recently been questioned by a radical view, wherein Rho binds to the elongating RNA polymerase (RNAP) prior to loading onto the mRNA. Using growth assays, microarray analyses and reporter-based transcription termination assays in vivo, we showed that slowing of the transcription elongation rate by using RNAP mutants (rpoB8 and rpoB3445) and growth of the strains in minimal medium suppressed the termination defects of five Rho mutants, three NusG mutants defective for Rho binding and the defects caused by two Rho inhibitors, Psu and bicyclomycin. These results established the existence of kinetically controlled steps in the in vivo Rho-dependent termination process and further reinforced the importance of ‘kinetic coupling’ between the two molecular motors, Rho and RNAP, and also argue strongly that the Rho translocation model is an accurate representation of the in vivo situation. Finally, these results indicated that one of the major roles of NusG in in vivo Rho-dependent termination is to enhance the speed of RNA release from the EC.

The BasS–BasR two-component system is known as an iron- and zinc-sensing transcription regulator in Escherichia coli, but so far only a few genes have been identified to be under the direct control of phosphorylated BasR. Using Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening, we have identified a total of at least 38 binding sites of phosphorylated BasR on the E. coli genome, and based on the BasR-binding sites, have predicted more than 20 novel targets of regulation. By DNase I footprint analysis for high-affinity BasR-binding sites, a direct repeat of a TTAAnnTT sequence was identified as the BasR box. Transcription regulation in vivo of the target genes was confirmed after Northern blot analysis of target gene mRNAs from both wild-type E. coli and an otherwise isogenic basR deletion mutant. The BasR regulon can be classified into three groups of genes: group 1 includes the genes for the formation and modification of membrane structure; group 2 includes genes for modulation of membrane functions; and group 3 includes genes for stress-response cell functions, including csgD, the master regulator of biofilm formation.

R-type and F-type pyocins are high-molecular-mass bacteriocins produced by Pseudomonas aeruginosa that resemble bacteriophage tails. They contain no head structures and no DNA, and are used as defence systems. In this report, we show that Pseudomonas fluorescens SF4c, a strain isolated from the wheat rhizosphere, produces a high-molecular-mass bacteriocin which inhibits the growth of closely related bacteria. A mutant deficient in production of this antimicrobial compound was obtained by transposon mutagenesis. Sequence analysis revealed that the transposon had disrupted a gene that we have named ptm, since it is homologous to that encoding phage tape-measure protein in P. fluorescens Pf0-1, a gene belonging to a prophage similar to phage-like pyocin from P. aeruginosa PAO1. In addition, we have identified genes from the SF4c pyocin cluster that encode a lytic system and regulatory genes. We constructed a non-polar ptm mutant of P. fluorescens SF4c. Heterologous complementation of this mutation restored the production of bacteriocin. Real-time PCR was used to analyse the expression of pyocin under different stress conditions. Bacteriocin was upregulated by mitomycin C, UV light and hydrogen peroxide, and was downregulated by saline stress. This report constitutes, to our knowledge, the first genetic characterization of a phage tail-like bacteriocin in a rhizosphere Pseudomonas strain.

Staphylococcus aureus is the leading cause of nosocomial infections and a major cause of community-acquired infections. Biofilm formation is a key virulence determinant in certain types of S. aureus infection, especially those involving inserted medical devices. We found in a previous study that the calcium chelators sodium citrate and EGTA inhibit biofilm formation in certain strains of S. aureus but actually augment biofilm formation in other strains. Even two closely related strains, Newman and 10833, exhibited strikingly different biofilm phenotypes in the presence of calcium chelators, in that biofilm formation was inhibited in Newman but augmented in 10833. We also found that the surface protein clumping factor B (ClfB) plays a role in this phenomenon. In this study, we confirm that ClfB is required for biofilm formation under calcium-depleted conditions. We investigated the post-translational regulation of ClfB-mediated biofilm formation and found evidence that both calcium and the protease aureolysin disrupt established ClfB-dependent biofilms. Finally, we investigated the genetic basis for the biofilm-negative phenotype in strain Newman versus the biofilm-positive phenotype in strain 10833 under calcium-depleted conditions and found that strain 10833 contains a deletion that results in a stop codon within the aureolysin gene (aur). When 10833 expressed Newman aur, surface-associated ClfB and the ability to form a biofilm in chelating conditions was lost. Thus, the positive effect of chelating agents on biofilm formation in certain strains can be explained by increased ClfB activity in the absence of calcium and the discrepancy in the response of strains 10833 and Newman can be explained by point mutations in aur. This study reveals a previously unknown, to our knowledge, role for ClfB in biofilm formation and underscores the potential for striking phenotypic variability resulting from minor differences in strain background.

Sulfolobus species have been developed as excellent model organisms to address fundamental questions of archaeal biology. Interesting patterns of natural variation among Sulfolobus islandicus strains have been identified through genome sequencing. Experimentally testing hypotheses about the biological causes and consequences of this natural variation requires genetic tools that apply to a diversity of strains. Previously, a genetic transformation system for S. islandicus was reported, in which overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene on the shuttle vector pSSR allowed the selection of transformants resistant to high concentrations of the thermostable antibiotic simvastatin. Here, we developed a novel gene knockout system based on simvastatin resistance. With this system, we created via homologous recombination an in-frame, markerless deletion of the intact S. islandicus M.16.4 pyrEF genes encoding orotidine-5′-monophosphate pyrophosphorylase (OPRTase) and orotidine-5′-monophosphate decarboxylase (OMPdecase), and a disruption of the lacS gene encoding β-galactosidase. Phenotypic analyses of the mutants revealed that the pyrEF deletion mutant lost the ability to synthesize uracil, and the lacS deletion mutants exhibited a white colour after X-Gal staining, demonstrating that the β-galactosidase function was inactivated. Our data demonstrate efficient tools to generate gene knockouts in a broad range of wild-type Sulfolobus strains.

Enterococci constitute a significant component of the lactic acid bacteria normally present in the intestinal microflora and include strains that produce bacteriocins. The genetic determinants for durancin GL in Enterococcus durans 41D were identified on the 8347 bp plasmid pDGL1 by plasmid curing experiments. pDGL1 contained nine putative ORFs, with ORF1 and ORF2 encoding plasmid replication proteins, and ORF3 and ORF6 showing high similarity to genes encoding mobilization proteins. The predicted protein encoded by ORF4 showed 74 % identity to BacA, a bacteriocin produced by Enterococcus faecalis. The deduced DurA protein contained the conserved motif YYGNG, suggesting that durancin GL is a typical subclass IIa bacteriocin. ORF5 was shown to share 85 % identity to the immunity protein BacB of Enterococcus faecalis. ORF9 displayed 87 % sequence identity to a conserved hypothetical protein of unknown function. To further clarify the minimum requirement for durancin GL production, a 547 bp fragment containing the durAB gene was fitted with the Streptococcus thermophilus P2201 promoter and then subcloned and heterologously expressed in S. thermophilus ST128. The result demonstrated that the cloned fragment contained all the genetic components required for durancin GL production.

Although Escherichia coli and Salmonella enterica serovar Typhimurium have a similar flagellar regulatory system, the response of flagellar synthesis to nutrient conditions is quite different between the two: that is, in low-nutrient conditions, flagellar synthesis is inhibited in Salmonella and enhanced in E. coli. In Salmonella, this inhibition is mediated by an anti-FlhD4C2 factor, YdiV, which is expressed in low-nutrient conditions and binds to FlhD4C2 to inhibit the expression of the class 2 flagellar genes. The fliZ gene encodes a repressor of the ydiV gene, and thus is required for efficient flagellar gene expression in low-nutrient conditions in Salmonella. In this study, we showed that the E. coli ydiV gene encodes a protein which inhibits motility and flagellar production when expressed from a multicopy plasmid. We showed further that E. coli YdiV binds to FlhD4C2 and inhibits its binding to the class 2 flagellar promoter. These results indicate that E. coli YdiV can also act as an anti-FlhD4C2 factor. However, although the ydiV gene was transcribed efficiently in E. coli cells, the intracellular level of the YdiV protein was extremely low due to its inefficient translation. Consistent with this, E. coli cells did not require FliZ for efficient motility development. This indicates that, unlike in Salmonella, the FliZ–YdiV regulatory system does not work in the nutritional control of flagellar gene expression in E. coli.

Bioinformatic approaches employed to analyse intergenic regions of Pseudomonas aeruginosa O1 (PAO1) for small RNAs (sRNAs) revealed a putative RNA gene encoded upstream of the nitrate assimilation operon nirBD–PA1779–cobA. Here, we show that this RNA, termed nitrogen assimilation leader A (NalA), represents the leader RNA of the nirBD–PA1779–cobA operon, and that nalA transcription is σ54- and NtrC-dependent. A PAO1 nalA deletion strain and a strain bearing a deletion in ORF PA1785 failed to grow on nitrate. PA1785 was identified as a homologue of the Azotobacter vinelandii nasT gene, the product of which is required for transcription of the A. vinelandii nitrite/nitrate reductase operon. Collectively, these studies reveal that transcriptional antitermination of the leader RNA NalA is required for expression of the PAO1 nitrate assimilation operon, and that this process is governed by conserved functions in PAO1 and A. vinelandii.

In common with other bacterial taxa, members of the genus Neisseria are classified using a range of phenotypic and biochemical approaches, which are not entirely satisfactory in assigning isolates to species groups. Recently, there has been increasing interest in using nucleotide sequences for bacterial typing and taxonomy, but to date, no broadly accepted alternative to conventional methods is available. Here, the taxonomic relationships of 55 representative members of the genus Neisseria have been analysed using whole-genome sequence data. As genetic material belonging to the accessory genome is widely shared among different taxa but not present in all isolates, this analysis indexed nucleotide sequence variation within sets of genes, specifically protein-coding genes that were present and directly comparable in all isolates. Variation in these genes identified seven species groups, which were robust to the choice of genes and phylogenetic clustering methods used. The groupings were largely, but not completely, congruent with current species designations, with some minor changes in nomenclature and the reassignment of a few isolates necessary. In particular, these data showed that isolates classified as Neisseria polysaccharea are polyphyletic and probably include more than one taxonomically distinct organism. The seven groups could be reliably and rapidly generated with sequence variation within the 53 ribosomal protein subunit (rps) genes, further demonstrating that ribosomal multilocus sequence typing (rMLST) is a practicable and powerful means of characterizing bacteria at all levels, from domain to strain.