- Volume 161, Issue 9, 2015

Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs. Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration.

Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5′-NTAA-3′ flanking the 3′ end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

Vibrio harveyi CAIM 1792 is a marine bacterial strain that causes mortality in farmed shrimp in north-west Mexico, and the identification of virulence genes in this strain is important for understanding its pathogenicity. The aim of this work was to compare the V. harveyi CAIM 1792 genome with related genome sequences to determine their phylogenic relationship and explore unique regions in silico that differentiate this strain from other V. harveyi strains. Twenty-one newly sequenced genomes were compared in silico against the CAIM 1792 genome at nucleotidic and predicted proteome levels. The proteome of CAIM 1792 had higher similarity to those of other V. harveyi strains (78 %) than to those of the other closely related species Vibrio owensii (67 %), Vibrio rotiferianus (63 %) and Vibrio campbellii (59 %). Pan-genome ORFans trees showed the best fit with the accepted phylogeny based on DNA–DNA hybridization and multi-locus sequence analysis of 11 concatenated housekeeping genes. SNP analysis clustered 34/38 genomes within their accepted species. The pangenomic and SNP trees showed that V. harveyi is the most conserved of the four species studied and V. campbellii may be divided into at least three subspecies, supported by intergenomic distance analysis. blastp atlases were created to identify unique regions among the genomes most related to V. harveyi CAIM 1792; these regions included genes encoding glycosyltransferases, specific type restriction modification systems and a transcriptional regulator, LysR, reported to be involved in virulence, metabolism, quorum sensing and motility.

Escherichia coli strains are normally identified by the combination of their O and H (and sometimes K) antigens, and serotyping based on the antigens is believed to be crucial for clinical detection and epidemiological investigation. Two E. coli strains, G5413 and G5287, were isolated from faecal samples of female patients with diarrhoea and were not agglutinated with any antisera that cover the well-known O serogroups of E. coli. We elucidated the O-polysaccharide (OPS) structures and analysed the O-antigen gene clusters of these bacteria. The OPS structure of G5413 established by monosaccharide analysis and NMR spectroscopy was found to be unique amongst known bacterial polysaccharide structures. The O-antigen gene cluster of this strain was sequenced and did not match sequence data with any of the 184 O serogroups that have been recognized internationally. Gene functions were tentatively assigned and were appropriate to the OPS structure. Based on these data, we suggest G5413 as a candidate for a new E. coli O serogroup. Both the OPS structure and O-antigen gene cluster of G5287 were identical to those of E. coli L-19, a candidate for another new O serogroup characterized by us recently. Recognition of these two provisional O serogroups increases the number of known O-antigen forms of E. coli to 186.

Rhizobium etli aerobically respires with several terminal oxidases. The quinol oxidase (Cyo) encoded by cyoABCD is needed for efficient adaptation to low oxygen conditions and cyo transcription is upregulated at low oxygen. This study sought to determine how transcription of the cyo operon is regulated. The 5′ sequence upstream of cyo was analysed in silico and revealed putative binding sites for ActR of the ActSR two-component regulatory system. The expression of cyo was decreased in an actSR mutant regardless of the oxygen condition. As ActSR is known to be important for growth under low pH in another rhizobial species, the effect of growth medium pH on cyo expression was tested. As the pH of the media was incrementally decreased, cyo expression gradually increased in the WT, eventually reaching ∼10-fold higher levels at low pH (4.8) compared with neutral pH (7.0) conditions. This upregulation of cyo under decreasing pH conditions was eliminated in the actSR mutant. Both the actSR and cyo mutants had severe growth defects at low pH (4.8). Lastly, the actSR and cyo mutants had severe growth defects when grown in media treated with an iron chelator. Under these conditions, cyo was upregulated in the WT, whereas cyo was not induced in the actSR mutant. Altogether, the results indicated cyo expression is largely dependent on the ActSR two-component system. This study also demonstrated additional physiological roles for Cyo in R. etli CFN42, in which it is the preferred oxidase for growth under acidic and low iron conditions.

CbbR is a LysR-type transcriptional regulator that activates expression of the operons containing (cbb) genes that encode the CO2 fixation pathway enzymes in Ralstonia eutropha (Cupriavidus necator) under autotrophic growth conditions. The cbb operons are stringently downregulated during chemoheterotrophic growth on organic acids such as malate. CbbR constitutive proteins (CbbR*s), typically with single amino acid substitutions, were selected and isolated that activate expression of the cbb operons under chemoheterotrophic growth conditions. A large set of CbbR*s from all major domains of the CbbR molecule were identified, except for the DNA-binding domain. The level of gene expression conferred for many of these CbbR*s under autotrophic growth was greater than that conferred by wild-type CbbR. Several of these CbbR*s increase transcription two- to threefold more than wild-type CbbR. One particular CbbR*, a truncated protein, was useful in identifying the regions of CbbR that are necessary for transcriptional activation and, by logical extension, necessary for interaction with RNA polymerase. The reductive assimilation of carbon via CO2 fixation is an important step in the cost-effective production of useful biological compounds. Enhancing CO2 fixation in Ralstonia eutropha through greater transcriptional activation of the cbb operons could prove advantageous, and the use of CbbR*s is one way to enhance product formation.

The Gram-positive Corynebacterium glutamicum co-metabolizes most carbon sources such as the phosphotransferase system (PTS) sugar glucose and the non-PTS sugar maltose. Maltose is taken up via the ABC-transporter MusEFGK2I, and is further metabolized to glucose phosphate by amylomaltase MalQ, maltodextrin phosphorylase MalP, glucokinase Glk and phosophoglucomutase Pgm. Surprisingly, growth of C. glutamicum strains lacking the general PTS components EI or HPr was strongly impaired on the non-PTS sugar maltose. Complementation experiments showed that a functional PTS phosphorelay is required for optimal growth of C. glutamicum on maltose, implying its involvement in the control of maltose metabolism and/or uptake. To identify the target of this PTS-dependent control, transport measurements with 14C-labelled maltose, Northern blot analyses and enzyme assays were performed. The activities of the maltose transporter and enzymes MalQ, Pgm and GlK were not decreased in PTS-deficient C. glutamicum strains, which was corroborated by comparable transcript amounts of musE, musK and musG, as well as of malQ, in C. glutamicum ΔptsH and WT. By contrast, MalP activity was significantly reduced and only residual amounts of malP transcripts were detected in C. glutamicum ΔptsH when compared to WT. Promoter activity assays with the malP promoter in C. glutamicum ΔptsH and WT confirmed that malP transcription is reduced in the PTS-deficient strain. Taken together, we show here for what is to the best of our knowledge the first time a regulatory function of the PTS in C. glutamicum and identify malP transcription as its target.

It has repeatedly been shown that aryl-hydroxylating dioxygenases do not possess a very high substrate specificity. To gain more insight into this phenomenon, we examined two powerful biphenyl dioxygenases, the well-known wild-type enzyme from Burkholderia xenovorans LB400 (BphA-LB400) and a hybrid enzyme, based on a dioxygenase from Pseudomonas sp. B4-Magdeburg (BphA-B4h), for their abilities to dioxygenate a selection of eight biphenyl analogues in which the second aromatic ring was replaced by aliphatic as well as aliphatic/aromatic moieties, reflecting a variety of steric requirements. Interestingly, both enzymes were able to catalyse transformation of almost all of these compounds. While the products formed were identical, major differences were observed in transformation rates. In most cases, BphA-B4h proved to be a significantly more powerful catalyst than BphA-LB400. NMR characterization of the reaction products showed that the metabolite obtained from biphenylene underwent angular dioxygenation, whereas all other compounds were subject to lateral dioxygenation at ortho and meta carbons. Subsequent growth studies revealed that both dioxygenase source strains were able to utilize several of the biphenyl analogues as sole sources of carbon and energy. Therefore, prototype BphBCD enzymes of the biphenyl degradative pathway were examined for their ability to further catabolize the lateral dioxygenation products. All of the ortho- and meta-hydroxylated compounds were converted to acids, showing that this pathway is quite permissive, enabling catalysis of the turnover of a fairly wide variety of metabolites.

Streptococcus mutans in dental biofilms often faces life-threatening threats such as killing by antimicrobial molecules from competing species or from the host. The ability of S. mutans to cope with such threats is crucial for its survival and persistence in dental biofilms. By screening a transposon mutant library, we identified 11 transposon insertion mutants that were sensitive to bacitracin. Two of these mutants, XTn-01 and XTn-03, had an independent insertion in the same locus, SMU.244, which encoded a homologue of undecaprenyl pyrophosphate phosphatase (UppP). In this study, we describe the genetic and phenotypic characterization of SMU.244 in antibiotic resistance. The results revealed that deletion of SMU.244 results in a mutant (XTΔ244) that is highly sensitive to bacitracin, but confers more resistance to lactococcin G, a class IIb bacteriocin. Introduction of the intact SMU.244 into XTΔ244 in trans completely restores its resistance to bacitracin and the susceptibility to lactococcin G. The XTΔ244 was also defective in forming the WT biofilm, although its growth was not significantly affected. Using recombinant protein technology, we demonstrated that the SMU.244-encoded protein displays enzyme activity to catalyse dephosphorylation of the substrate. The lux transcriptional reporter assays showed that S. mutans maintains a moderate level of expression of SMU.244 in the absence of bacitracin, but bacitracin at sub-MICs can further induce its expression. We concluded that SMU.244 encodes an UppP protein that plays important roles in cell wall biosynthesis and bacitracin resistance in S. mutans. The results described here may further our understanding of the molecular mechanisms by which S. mutans copes with antibiotics such as bacitracin.

RsmA is a post-transcriptional RNA-binding protein that acts as a pleiotropic global regulator of mRNAs in the opportunistic pathogen Pseudomonas aeruginosa. Upon binding to its target, RsmA impedes the translation of the mRNA by the ribosome. The RsmA regulon affects over 500 genes, many of which have been identified as important in the pathogenicity of P. aeruginosa. Whilst the regulatory function of RsmA is relatively well characterized, the genetic regulation of rsmA itself at the transcriptional and translational levels remains poorly understood. Here, we show that RsmA is capable of self-regulation through an unorthodox mechanism. This regulation occurs via direct interaction of the protein with an RsmA-binding site located in the early portion of its coding sequence. To the best of our knowledge this is the first report of such an unusual regulation in pseudomonads.